Magdalena Ciechanowska

Neuroendocrine regulation of GnRH release and expression of GnRH and GnRH receptor genes in the hypothalamus-pituitary unit in different physiological states

This review is focused on the relationship between neuroendocrine regulation of GnRH/LH secretion and the expression of GnRH and GnRH receptor (GnRHR) genes in the hypothalamic-pituitary unit during different physiological states of animals and under stress. Moreover, the involvement of hypothalamic GABA-ergic, Beta-endorphinergic, CRH-ergic, noradrenergic, dopaminergic and GnRH-ergic systems in the regulation of expression of the GnRH and GnRHR genes as well as secretion of GnRH/LH is analyzed. It appears that the neural mechanisms controlling GnRH gene expression in different physiological states may be distinct from those regulating GnRH/LH release. The hypothalamic GnRHR gene is probably located in different neural systems and may act in a specific way on GnRH gene expression and GnRH release.

Changes in the GnRH mRNA and GnRH receptor (GnRH-R) mRNA levels in the hypothalamic-anterior pituitary unit of anestrous ewes after infusion of GnRH into the third cerebral ventricle.

In the present paper the role of GnRH in the ultrashort loop of the negative feedback action on GnRH secretion was evaluated on the molecular level by the Real-time PCR technique. Specifically, the effect of GnRH infused into the third cerebral ventricle on the expression of GnRH and GnRH receptor (GnRH-R) genes was analyzed in the hypothalamic-pituitary unit of anestrous ewes. GnRH did not significantly affect GnRH mRNA levels in the preoptic/anterior hypothalamic area but drastically increased its level in the ventromedial hypothalamus. In addition, GnRH infusion augmented GnRH-R mRNA level in the entire hypothalamus. In the GnRH-treated animals, anterior pituitary GnRH-R mRNA level and plasma LH concentration were also elevated. The changes in GnRH mRNA and GnRH-R mRNA levels in the hypothalamus in response to treatment with GnRH suggest that GnRH acts differently on the stability of these transcripts. On the basis of presented results it seems that GnRH may affect GnRH and GnRH-R biosynthesis and, consequently, GnRH/LH release.

Effects of corticotropin-releasing hormone and its antagonist on the gene expression of gonadotrophin-releasing hormone (GnRH) and GnRH receptor in the hypothalamus and anterior pituitary gland of follicular phase ewes

There is no information in the literature regarding the effect of corticotropin-releasing hormone (CRH) on genes encoding gonadotrophin-releasing hormone (GnRH) and the GnRH receptor (GnRHR) in the hypothalamus or on GnRHR gene expression in the pituitary gland in vivo. Thus, the aim of the present study was to investigate, in follicular phase ewes, the effects of prolonged, intermittent infusion of small doses of CRH or its antagonist (α-helical CRH 9-41; CRH-A) into the third cerebral ventricle on GnRH mRNA and GnRHR mRNA levels in the hypothalamo-pituitary unit and on LH secretion. Stimulation or inhibition of CRH receptors significantly decreased or increased GnRH gene expression in the hypothalamus, respectively, and led to different responses in GnRHR gene expression in discrete hypothalamic areas. For example, CRH increased GnRHR gene expression in the preoptic area, but decreased it in the hypothalamus/stalk median eminence and in the anterior pituitary gland. In addition, CRH decreased LH secretion. Blockade of CRH receptors had the opposite effect on GnRHR gene expression. The results suggest that activation of CRH receptors in the hypothalamus of follicular phase ewes can modulate the biosynthesis and release of GnRH through complex changes in the expression of GnRH and GnRHR genes in the hypothalamo-anterior pituitary unit.

Effects of GABAA receptor modulation on the expression of GnRH gene and GnRH receptor (GnRH-R) gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland of follicular-phase ewes.

The effect of prolonged, intermittent infusion of GABA(A) receptor agonist (muscimol) or GABA(A) receptor antagonist (bicuculline) into the third cerebral ventricle on the expression of GnRH gene and GnRH-R gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland was examined in follicular-phase ewes by real-time PCR. The activation or inhibition of GABA(A) receptors in the hypothalamus decreased or increased the expression of GnRH and GnRH-R genes and LH secretion, respectively. The present results indicate that the GABAergic system in the hypothalamus of follicular-phase ewes may suppress, via hypothalamic GABA(A) receptors, the expression of GnRH and GnRH-R genes in this structure. The decrease or increase of GnRH-R mRNA in the anterior pituitary gland and LH secretion in the muscimol- or bicuculline-treated ewes, respectively, is probably a consequence of parallel changes in the release of GnRH from the hypothalamus activating GnRH-R gene expression. It is suggested that GABA acting through the GABA(A) receptor mechanism on the expression of GnRH gene and GnRH-R gene in the hypothalamus may be involved in two processes: the biosynthesis of GnRH and the release of this neurohormone in the hypothalamus.

Effect of short-term and prolonged stress on the biosynthesis of gonadotropin-releasing hormone (GnRH) and GnRH receptor (GnRHR) in the hypothalamus and GnRHR in the pituitary of ewes during various physiological states

Using an ELISA assay, the levels of GnRH and GnRHR were analysed in the preoptic area (POA), anterior (AH) and ventromedial hypothalamus (VM), stalk/median eminence (SME); and GnRHR in the anterior pituitary gland (AP) of non-breeding and breeding sheep subjected to short-term or prolonged stress. The ELISA study was supplemented with an analysis of plasma LH concentration. Short-term footshock stimulation significantly increased GnRH levels in hypothalamus in both seasons. Prolonged stress elevated or decreased GnRH concentrations in the POA and the VM, respectively during anoestrus, and lowered GnRH amount in the POA-hypothalamus of follicular-phase sheep. An up-regulation of GnRHR levels was noted in both, anoestrous and follicular-phase animals. In the non-breeding period, a prolonged stress procedure increased GnRHR biosynthesis in the VM and decreased it in the SME and AP, while in the breeding time the quantities of GnRHR were significantly lower in the whole hypothalamus. In follicular-phase ewes the fluctuations of GnRH and GnRHR levels under short-term and prolonged stress were reflected in the changes of LH secretion, suggesting the existence of a direct relationship between GnRH and GnRH-R biosynthesis and GnRH/LH release in this period. The study showed that stress was capable of modulating the biosynthesis of GnRH and GnRHR; the pattern of changes was dependent upon the animals physiological state and on the time course of stressor application. The obtained results indicate that the disturbances of gonadotropin secretion under stress conditions in sheep may be due to a dysfunction of GnRH and GnRHR biosynthetic pathways.

The influence of dopaminergic system inhibition on biosynthesis of gonadotrophin-releasing hormone (GnRH) and GnRH receptor in anoestrous sheep; hierarchical role of kisspeptin and RFamide-related peptide-3 (RFRP-3)

This study aimed to explain how prolonged inhibition of central dopaminergic activity affects the cellular processes governing gonadotrophin-releasing hormone (GnRH) and LH secretion in anoestrous sheep. For this purpose, the study included two experimental approaches: first, we investigated the effect of infusion of sulpiride, a dopaminergic D2 receptor antagonist (D2R), on GnRH and GnRH receptor (GnRHR) biosynthesis in the hypothalamus and on GnRHR in the anterior pituitary using an immunoassay. This analysis was supplemented by analysis of plasma LH levels by radioimmunoassay. Second, we used real-time polymerase chain reaction to analyse the influence of sulpiride on the levels of kisspeptin (Kiss1) mRNA in the preoptic area and ventromedial hypothalamus including arcuate nucleus (VMH/ARC), and RFamide-related peptide-3 (RFRP-3) mRNA in the paraventricular nucleus (PVN) and dorsomedial hypothalamic nucleus. Sulpiride significantly increased plasma LH concentration and the levels of GnRH and GnRHR in the hypothalamic-pituitary unit. The abolition of dopaminergic activity resulted in a significant increase in transcript level of Kiss1 in VMH/ARC and a decrease of RFRP-3 in PVN. The study demonstrates that dopaminergic neurotransmission through D2R is involved in the regulatory pathways of GnRH and GnRHR biosynthesis in the hypothalamic-pituitary unit of anoestrous sheep, conceivably via mechanisms in which Kiss1 and RFRP-3 participate.