Tumor Necrosis Factor α Impairs Kisspeptin Signaling in Human Gonadotropin-Releasing Hormone Primary Neurons

CONTEXT: Previous studies have suggested that inflammatory pathways may impair central regulatory networks involving gonadotropin-releasing hormone (GnRH) neuron activity. Studies in humans are strongly hampered by the lack of human GnRH neuron cell lines. OBJECTIVE: To establish an in vitro model of GnRH neurons of human origin and analyze the effects of proinflammatory cytokines on their biological properties. Design, samples and treatments: The primary human fetal hypothalamic (hfHypo) cell cultures were isolated from brain of three 12 week-old fetuses. Responsiveness to kisspeptin (0.1nM-1μ M), the main physiological regulator of GnRH neurons, was evaluated for biological characterization of hfHypo cells. Tumor necrosis factor alpha (TNFα, 10 ng/ml, 5h and 24h) was used as pro-inflammatory stimulus. MAIN OUTCOME MEASURES: Expression of specific GnRH neuron markers by quantitative RT-PCR, flow cytometry and immunocytochemistry analyses; GnRH releasing ability and electrophysiological changes in response to kisspeptin stimulation. RESULTS: The primary hfHypo cell cultures were characterized by a high percentage of GnRH-positive cells (80%), abundantly expressing the kisspeptin receptor (KISS1R) and able to release GnRH in response to kisspeptin. Moreover, kisspeptin induced changes in electrophysiological membrane properties. TNF|ga exposure determined a specific inflammatory intracellular signaling and significantly reduced GnRH secretion, KISS1R expression and kisspeptin-induced depolarizing effect. In addition, hfHypo cells possessed a primary cilium, whose assembly was significantly inhibited by TNF|ga treatment. CONCLUSION: The hfHypo cells represent a novel tool for in vitro investigations on human GnRH neuron biology. TNFΑ may directly affect GnRH neuron function by interfering with KISS1R expression and ciliogenesis, thereby impairing kisspeptin signaling.