Mechanisms of central neural regulation of GnRH/LH secretion under short and prolonged stress in the hypothalamic-pituitary unit of ewes in different stages of reproduction

AIM: The aim of this study was (1) to analyze the effects of short and prolonged stress (footshock stimulation) on the biosynthesis of GnRH and GnRHR proteins in the hypothalamus-pituitary region and on luteinizing hormone (LH) secretion in anestrous and follicular phase ewes (2) to determine whether applied models of physical stress influence mRNA expression of kisspeptin (kiss1) and RFamide-related peptide-3 (RFRP-3), two central regulators of the mammalian reproductive axis. METHODS: The levels of the GnRH and GnRHR proteins were analyzed in selected tissue of the hypothalamus-anterior pituitary unit using an enzyme-linked immunoabsorbent assay (ELISA). Plasma LH concentration was measured by a double-antibody radioimmunoassay. To determine the transcript levels of kiss 1 and RFRP-3 in the preoptic area-hypothalamus region, Real-time PCR with SYBR Green dye was applied. RESULTS: Stress changed drastically the biosynthesis of GnRH and GnRHR, as well as the transcriptional activities of genes encoding kiss 1 and RFRP3 neuropeptides. The pattern of these changes was dependent upon physiological state of animal and on the time course of stressor application. The fluctuations of GnRH and GnRH-R protein levels under short or prolonged stress stimuli were associated with similar changes in LH secretion, thus suggesting the existence of a direct relationship between GnRH and GnRH-R biosynthesis and GnRH/LH release. CONCLUSIONS: The results indicate that disturbances of gonadotropin secretion under stress condition in sheep may be due to dysfunction of the hypothalamus/pituitary GnRH/GnRHR system. It can not be excluded that interaction between kiss 1 and RFRP3 neuronal networks with GnRH cells may also play a critical role in the transduction of stress-induced changes in the activity of hypothalamic-pituitary-gonadal axis. However, a detailed explanation of this phenomenon requires further research.