Tissue-Slice Organ-on-Chip Culture of Hypothalamic and Pituitary of Lambs─The Role of Phoenixin-20 as a Modulator of Gonadotrophic Axis

This study aimed to reconstruct the hypothalamic-pituitary axis using an organ-on-a-chip (OOC) model and to evaluate the modulatory role of phoenixin-20 (PNX) in the regulation of the gonadotrophic axis in sheep. Sixteen female Polish Merino lambs were used as tissue donors to create microfluidic chips containing paired hypothalamic and pituitary slices connected via perfused channels. This system enabled continuous medium flow and maintenance of functional neuroendocrine interactions under ex vivo conditions. The OOC platform was used to analyze changes in the expression of gonadotropin-releasing hormone (GnRH), kisspeptin (Kiss), neurokinin B (NKB), and prodynorphin (pDYN) in the hypothalamus, as well as luteinizing hormone (LH) and follicle-stimulating hormone (FSH) expression and secretion in the pituitary. PNX treatment significantly increased hypothalamic GnRH expression, while the blockade of neuropeptide Y receptors (NPY1R and NPY5R) diminished this response, suggesting that PNX effects are at least partially mediated through NPY-dependent pathways. Moreover, PNX altered the transcription of Kiss, NKB, and pDYN, key components of the GnRH pulse generator, and modulated LHβ mRNA expression in the pituitary. Changes in the LH and FSH concentrations further supported a receptor-specific mechanism of PNX action. The developed hypothalamo-pituitary OOC model proved valuable for studying neuroendocrine control of reproduction. This system offers a physiologically relevant and ethically sustainable alternative to <i>in vivo</i> experiments, enabling precise investigations of molecular and hormonal mechanisms within the gonadotrophic axis.