Erica Sarchielli1, Paolo Comeglio2, Roberta Squecco3, Lara Ballerini4, Tommaso Mello5, Giulia Guarnieri1, Eglantina Idrizaj3, Benedetta Mazzanti4, Linda Vignozzi2, Pasquale Gallina6, Mario Maggi2,7, Gabriella B Vannelli1, Annamaria Morelli1. 1. Section of Human Anatomy and Histology. 2. Sexual Medicine and Andrology Unit and. 3. Section of Physiological Sciences, and. 4. Cell Therapy and Transfusion Medicine Unit, Department of Experimental and Clinical Medicine, University of Florence, 50134 Florence, Italy. 5. Gastroenterology Unit, Department of Experimental and Clinical Biomedical Sciences "Mario Serio," University of Florence, 50134 Florence, Italy. 6. Neurosurgery School of Tuscany, Department of Surgery and Translational Medicine, University of Florence, 50139 Florence, Italy; and. 7. Istituto Nazionale Biostrutture e Biosistemi, 00136 Rome, Italy.
Abstract
Context: Inflammatory pathways may impair central regulatory networks involving gonadotropin-releasing hormone (GnRH) neuron activity. Studies in humans are limited by the lack of human GnRH neuron cell lines. Objective: To establish an in vitro model of human GnRH neurons and analyze the effects of proinflammatory cytokines. Design: The primary human fetal hypothalamic cells (hfHypo) were isolated from 12-week-old fetuses. Responsiveness to kisspeptin, the main GnRH neurons' physiological regulator, was evaluated for biological characterization. Tumor necrosis factor alpha (TNF-α) was used as a proinflammatory stimulus. Main Outcome Measures: Expression of specific GnRH neuron markers by quantitative reverse transcription-polymerase chain reaction, flow cytometry, and immunocytochemistry analyses; and GnRH-releasing ability and electrophysiological changes in response to kisspeptin. Results: The primary hfHypo showed a high percentage of GnRH-positive cells (80%), expressing a functional kisspeptin receptor (KISS1R) and able to release GnRH in response to kisspeptin. TNF-α exposure determined a specific inflammatory intracellular signaling and reduced GnRH secretion, KISS1R expression, and kisspeptin-induced depolarizing effect. Moreover, hfHypo possessed a primary cilium, whose assembly was inhibited by TNF-α. Conclusion: The hfHypo cells represent a novel tool for investigations on human GnRH neuron biology. TNF-α directly affects GnRH neuron function by interfering with KISS1R expression and ciliogenesis, thereby impairing kisspeptin signaling.
Context: Inflammatory pathways may impair central regulatory networks involving gonadotropin-releasing hormone (GnRH) neuron activity. Studies in humans are limited by the lack of humanGnRH neuron cell lines. Objective: To establish an in vitro model of humanGnRH neurons and analyze the effects of proinflammatory cytokines. Design: The primary human fetal hypothalamic cells (hfHypo) were isolated from 12-week-old fetuses. Responsiveness to kisspeptin, the main GnRH neurons' physiological regulator, was evaluated for biological characterization. Tumor necrosis factor alpha (TNF-α) was used as a proinflammatory stimulus. Main Outcome Measures: Expression of specific GnRH neuron markers by quantitative reverse transcription-polymerase chain reaction, flow cytometry, and immunocytochemistry analyses; and GnRH-releasing ability and electrophysiological changes in response to kisspeptin. Results: The primary hfHypo showed a high percentage of GnRH-positive cells (80%), expressing a functional kisspeptin receptor (KISS1R) and able to release GnRH in response to kisspeptin. TNF-α exposure determined a specific inflammatory intracellular signaling and reduced GnRH secretion, KISS1R expression, and kisspeptin-induced depolarizing effect. Moreover, hfHypo possessed a primary cilium, whose assembly was inhibited by TNF-α. Conclusion: The hfHypo cells represent a novel tool for investigations on humanGnRH neuron biology. TNF-α directly affects GnRH neuron function by interfering with KISS1R expression and ciliogenesis, thereby impairing kisspeptin signaling.
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