Development And Regulation Of The Gonadotropin Releasing Hormone System

对生殖至关重要的GnRH神经元来自鼻腔，并迁移到大脑，在那里它们成为下丘脑-垂体-性腺轴的组成部分。我们在正常/转基因动物和两种体外模型系统鼻外植体和GnRH衍生细胞系中研究GnRH神经元分化、迁移和轴突靶向的机制。利用这些动物和体外模型，我们的工作还解决了GnRH神经元中调节（内在和跨突触）GnRH基因表达、肽合成和分泌的机制。多种方法用于识别和理解在引导GnRH神经元到达其在中枢神经系统中的最终位置中起作用的众多分子和因素。这些包括从迁移和非迁移细胞中获得的文库的差异筛选，在迁移路线上关键位置差异表达的分子的检查，敲除小鼠GnRH系统发育的形态学检查，以及体外分子扰动和随后监测GnRH神经元运动。随着GnRH神经元的迁移，它们也成熟了，这两个过程实际上可能是有联系的。为了研究GnRH神经元的成熟，我们使用钙成像、电生理和生化方法来检测GnRH神经元的活性和肽分泌。此外，我们还与实验室合作，对生殖功能障碍患者进行人类基因筛查。一旦发现突变，我们分析小鼠的表达模式，并进行生物学分析，以确定突变基因对GnRH发育的影响。

GnRH neurons, critical for reproduction, are derived from the nasal placode and migrate into the brain where they become integral members of the hypothalamic-pituitary-gonadal axis. We study mechanism(s) underlying GnRH neuronal differentiation, migration and axonal targeting in normal/transgenic animals, and two in vitro model systems nasal explants and GnRH derived cell lines. Using these animal and in vitro models, our work also addresses the mechanisms regulating (intrinsic and trans-synaptic) GnRH gene expression, peptide synthesis and secretion in GnRH neurons. Multiple approaches are used to identify and understand the multitude of molecules and factors which play a role in directing the GnRH neurons to their final location in the CNS. These include differential screening of libraries obtained from migrating versus non-migrating cells, examination of molecules differentially expressed at key locations along the migratory route, morphological examination of the development of the GnRH system in knockout mice, and perturbation of molecules in vitro and subsequent monitoring of GnRH neuronal movement. As GnRH neurons migrate, they also mature, and the two processes may in fact be linked. To investigate the maturation of GnRH neurons, we use calcium imaging, electrophysiology and biochemical measures to examine GnRH neuronal activity and peptide secretion. In addition, we collaborate with labs performing human genetic screening of patients with reproductive dysfunction. Once a mutation is identified, we analyze the expression pattern in mice and perform biological assays to determine the outcome of the mutated gene on GnRH development. Over the past year, 2 primary articles have been published. The first article focuses on the development of the GnRH system, analyzing mutations identified in patients with delayed or absent puberty (Cho-H-J, Gurbuz F, Stamou M, Kotan LD, Farmer SM, Can S, Tompkins MF, Mamadova J, Altincik SA, Gokce C, Catli G, Bugrul F, Bartlett K, Turan I, Balasubramanian R, Yuksel B, Seminara SB, Wray S, Topaloglu AK. POU6F2 mutation in humans with pubertal failure alters GnRH transcript expression. Frontiers in Endocrinology, 2023). Idiopathic hypogonadotropic hypogonadism (IHH) is characterized by the absence of pubertal development and subsequent impaired fertility often due to gonadotropin-releasing hormone (GnRH) deficits. Exome sequencing of two independent cohorts of IHH patients identified 12 rare missense variants in POU6F2 in 15 patients. POU6F2 encodes two distinct isoforms. In the adult mouse, expression of both isoform1 and isoform2 was detected in the brain, pituitary, and gonads. However, only isoform1 was detected in mouse primary GnRH cells and three immortalized GnRH cell lines, two mouse and one human. To date, the function of isoform2 has been verified as a transcription factor, while the function of isoform1 has been unknown. In the present report, bioinformatics and cell assays on a human-derived GnRH cell line reveal a novel function for isoform1, demonstrating it can act as a transcriptional regulator, decreasing GNRH1 expression. In addition, the impact of the two most prevalent POU6F2 variants, identified in five IHH patients, that were located at/or close to the DNA-binding domain was examined. Notably, one of these mutations prevented the repression of GnRH transcripts by isoform1. Normally, GnRH transcription increases as GnRH cells mature as they near migrate into the brain. Augmentation earlier during development can disrupt normal GnRH cell migration, consistent with some POU6F2 variants contributing to the IHH pathogenesis. The second article (Shostak D, Constantin S, Flannery J, Wray S. Acetylcholine regulation of GnRH neuronal activity: A circuit in the medial septum, Frontiers in Endocrinology, 2023) focuses on regulation of GnRH neurons by acetylcholine (ACh). While it is known that ACh influences GnRH secretion, whether the effect is direct or indirect, and the specific ACh receptor (AChR) subtype(s) involved was unclear. In this report we determined 1) whether ACh can modulate GnRH cellular activity and 2) a source of ACh afferents contacting GnRH neurons. Calcium imaging was used to assay GnRH neuronal activity. With GABAergic and glutamatergic transmission blocked, subtype-specific AChR agonists and antagonists were applied to identify direct regulation of GnRH neurons. ACh and nicotine caused a rise in calcium that declined gradually back to baseline after 5-6 min. This response was mimicked by an alpha3-specific agonist. In contrast, muscarine inhibited GnRH calcium oscillations, and blocking M2 and M4 together prevented this inhibition. Labeling for choline acetyltransferase (ChAT) and GnRH revealed ChAT fibers contacting GnRH neurons, primarily in the medial septum (MS), and in greater number in females than males. ChAT positive cells in the MS are known to express p75NGFRs. Labeling for p75NGFR, ChAT and GnRH indicated that ChAT fibers contacting GnRH cells originate from cholinergic cells within these same rostral areas. Together, these results indicate that cholinergic cells in septal areas can directly regulate GnRH neurons.