Circadian Regulation of Gonadotropin-Releasing Hormone

描述(申请人提供)：正常的哺乳动物生殖依赖于下丘脑的促性腺激素释放激素(GnRH)的脉动分泌，而雌性的排卵需要GnRH的强劲激增。虽然众所周知，但目前尚不清楚这两种模式的肽释放时机背后的细胞和分子机制。虽然以前的研究表明24小时生物钟与生殖激素分泌的两种机制都有关，但目前尚不清楚生物钟如何在GnRH神经元内作用于调节GnRH同步分泌的幅度或频率来调节生殖。在之前的资助阶段，使用分泌GnRH的GT1-7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GnRH的分泌，可能导致生殖异常。此外，下面描述的初步研究表明，GnRH特异性时钟干扰改变了体内的生殖能力，内源性振荡器可能通过潜在地改变能够改变GnRH细胞兴奋性的调节器的时间模式，在传递雌激素对GnRH神经元的正反馈效应中发挥作用，从而导致释放浪涌所需的神经元活动改变。为了进一步研究这一点，以下建议将1)评估两个新的转基因小鼠模型，以确定多个下丘脑分子时钟在构建体内正常繁殖所需的GnRH释放模式中的各自贡献；2)探讨内源性GnRH时钟对雌激素刺激的峰分泌的需求和神经元活性的变化，并在体外和体内进行评估；3)检测雌激素对两个重要的神经调节剂，Kiss-1受体Gpr54和体外GnRH神经元中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参与。这项建议的结果解决了有关GnRH“脉冲发生器”的性质以及细胞-细胞和荷尔蒙交流如何改变分泌模式以产生排卵前激增的基本问题。此外，这些研究还提供了对内分泌神经分泌更广泛机制的洞察，并通过探索转录振荡如何控制同步的多细胞事件来调控众多生物过程，甚至协调复杂的行为，从而推动昼夜生物学的发展。潜在的应用可能为治疗由下丘脑神经分泌功能障碍引起的一系列生殖生理疾病带来新的方向，包括多囊卵巢综合征(PCOS)和原发性特发性性腺功能减退，这两种疾病都与非典型激素释放模式有关。 公共卫生相关性：这项提案产生的结果将解决基本问题，即细胞-细胞交流如何改变生殖神经分泌模式，以产生排卵所需的激素激增。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，调节众多生物过程，甚至协调复杂的行为，从而推动昼夜生物场的发展。潜在的应用可能为治疗由下丘脑神经分泌功能障碍引起的一系列生殖生理疾病带来新的方向，包括多囊卵巢综合征(PCOS)和原发性特发性性腺功能减退，这两种疾病都与非典型激素释放模式有关。

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.