Circadian Clock Regulation of Pulsatile GnRH Secretion

描述（由申请人提供）： 虽然人们早就知道，正常的哺乳动物生殖取决于下丘脑的促性腺激素释放激素（GnRH）的脉冲式分泌，但目前还不清楚这种异常定时脉冲释放的核心是什么细胞和分子机制。此外，虽然以前的研究表明24小时生物钟控制生殖激素分泌，但仍然不清楚昼夜节律钟如何调节同步GnRH分泌释放的幅度或频率以调节生殖。在这个实验室的初步工作，最近提交出版，表明不仅是所有的分子时钟组件，如mPer 1，mPer 2，和时钟存在于GnRH分泌的GT 1 -7细胞系，但转录mPer 1和mPer 2振荡在这些培养的细胞与昼夜节律周期。引人注目的是，在灌流的GT 1 -7细胞的时钟扰动，通过瞬时转染的显性负时钟，破坏正常的分泌脉冲模式，这表明细胞内的昼夜节律钟GnRH神经元内可能发挥调节分泌。为了进一步研究这一点，以下建议将尝试1）通过检查转录和分泌效应来确定分子钟对GnRH的调节程度; 2）研究仅在体内GnRH神经元中的细胞特异性时钟功能破坏如何影响转基因小鼠的生育力; 3）通过检查时钟基因振荡和膜动力学的实时变化，剖析将时钟振荡与定时GnRH脉冲分泌联系起来的细胞内和细胞间机制。该提案的结果有可能回答有关GnRH“脉冲发生器”性质的许多基本问题，提供对内分泌神经分泌广泛机制的见解，并允许候选人完全发展成为能够最终成为生殖神经生物学领导者的主要研究者。此外，这些研究还可以推进昼夜节律生物学领域，最终证明分子水平上的转录本振荡如何控制多细胞和组织水平上的同步事件，以调节从细胞代谢到胞吐作用的许多生物过程，甚至协调一系列复杂的行为。潜在的应用可能会导致新的方向，在治疗一系列的生理疾病，导致下丘脑神经分泌功能障碍，如多囊卵巢综合征和原发性特发性性腺功能减退症。

DESCRIPTION (provided by applicant): Whereas it has long been known that normal mammalian reproduction depends upon the pulsatile secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus, it is yet unclear what cellular and molecular mechanisms lie at the heart of this exceptionally timed pulse release. Additionally, while previous studies have implicated the 24 hour biological clock in the control of reproductive hormone secretion, it remains unknown how the circadian clock might modulate the amplitude or frequency of synchronous GnRH secretory release in order to regulate reproduction. Preliminary work in this laboratory, recently submitted for publication, indicates that not only are all molecular clock components, such as mPer1, mPer2, and clock present in the GnRH-secreting GT1-7 cell line, but that transcripts of mPer1 and mPer2 oscillate in these cultured cells with a circadian period. Strikingly, perturbation of the clock in perifused GT1-7 cells, via transient transfection of a dominant negative clock, disrupts normal secretory pulse patterns, suggesting that an intracellular circadian clock within GnRH neurons may function to modulate secretion. To investigate this further, the following proposal will attempt to 1) determine the extent of regulation of GnRH by the molecular clock, by examining both transcriptional and secretory effects; 2) investigate how cell-specific disruption of clock function only in GnRH neurons in vivo impacts fertility in transgenic mice; 3) dissect inter- and intracellular mechanisms linking clock oscillation to timed GnRH pulsatile secretion, by examining real-time changes in both clock gene oscillations and membrane kinetics. Results from this proposal have the potential to answer many fundamental questions regarding the nature of the GnRH "pulse generator," provide insight into broad mechanisms of endocrine neurosecretion, and allow for the candidate to fully develop into a principal investigator capable of eventually becoming a leader in reproductive neurobiology. Additionally, these studies could also advance the field of circadian biology, by ultimately demonstrating how oscillation of transcripts at the molecular level can control synchronous events at the multi-cellular and tissue level, in order to regulate numerous biological processes, from cellular metabolism to exocytosis, and even to orchestrate complex series of behaviors. Potential applications could lead to new directions in treating a range of physiological disorders that result from malfunction of hypothalamic neurosecretion, such as polycystic ovarian syndrome and primary ideopathic hypogonadism.