Does kisspeptin participate in GABA-mediated modulation of GnRH and GnRH receptor biosynthesis in the hypothalamic-pituitary unit of follicular-phase ewes?

BACKGROUND: The inverse relationship between GnRH transcript level and GABA neurons activity has suggested that GABA at the hypothalamic level may exert a suppressive effect on subsequent steps of the GnRH biosynthesis. In the present study, we analyzed the effects of GABA type A receptor agonist (muscimol) or antagonist (bicuculline) on molecular mechanisms governing GnRH/LH secretion in follicular-phase sheep. METHODS: ELISA technique was used to investigate the effects of muscimol and/or bicuculline on levels of post-translational products of genes encoding GnRH ligand and GnRH receptor (GnRHR) in the preoptic area (POA), anterior (AH) and ventromedial (VMH) hypothalamus, stalk/median eminence (SME), and GnRHR in the anterior pituitary (AP). Real-time PCR was chosen for determination of the effect of drugs on kisspeptin (Kiss 1) mRNA level in POA and VMH including arcuate nucleus (VMH/ARC), and on Kiss1 receptor (Kiss1r) mRNA abundance in POA-hypothalamic structures. These analyses were supplemented by RIA method for measurement of plasma LH concentration. RESULTS: The study demonstrated that muscimol and bicuculline significantly decreased or increased GnRH biosynthesis in all analyzed structures, respectively, and led to analogous changes in plasma LH concentration. Similar muscimol- and bicuculline-related alterations were observed in levels of GnRHR. However, the expression of Kiss 1 and Kiss1r mRNAs in selected POA-hypothalamic areas of either muscimol- and bicuculline-treated animals remained unaltered. CONCLUSIONS: Our data suggest that GABAergic neurotransmission is involved in the regulatory pathways of GnRH/GnRHR biosynthesis and then GnRH/LH release in follicular-phase sheep conceivably via indirect mechanisms that exclude involvement of Kiss 1 neurons.

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.

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.

Effects of GABA(A) 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.

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.

Expression of the GnRH and GnRH receptor (GnRH-R) genes in the hypothalamus and of the GnRH-R gene in the anterior pituitary gland of anestrous and luteal phase ewes.

Data exists showing that seasonal changes in the innervations of GnRH cells in the hypothalamus and functions of some neural systems affecting GnRH neurons are associated with GnRH release in ewes. Consequently, we put the question as to how the expression of GnRH gene and GnRH-R gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland is reflected with LH secretion in anestrous and luteal phase ewes. Analysis of GnRH gene expression by RT-PCR in anestrous ewes indicated comparable levels of GnRH mRNA in the preoptic area, anterior and ventromedial hypothalamus. GnRH-R mRNA at different concentrations was found throughout the preoptic area, anterior and ventromedial hypothalamus, stalk/median eminence and in the anterior pituitary gland. The highest GnRH-R mRNA levels were detected in the stalk/median eminence and in the anterior pituitary gland. During the luteal phase of the estrous cycle in ewes, the levels of GnRH mRNA and GnRH-R mRNA in all structures were significantly higher than in anestrous ewes. Also LH concentrations in blood plasma of luteal phase ewes were significantly higher than those of anestrous ewes. In conclusion, results from this study suggest that low expression of the GnRH and GnRH-R genes in the hypothalamus and of the GnRH-R gene in the anterior pituitary gland, amongst others, may be responsible for a decrease in LH secretion and the anovulatory state in ewes during the long photoperiod.

Effect of stress on the expression of GnRH and GnRH receptor (GnRH-R) genes in the preoptic area-hypothalamus and GnRH-R gene in the stalk/median eminence and anterior pituitary gland in ewes during follicular phase of the estrous cycle.

The RT-PCR (reverse transcription polymerase chain reaction) technique was used to analyze GnRH mRNA and GnRH-R mRNA in the preoptic area, anterior and ventromedial hypothalamus, and GnRH-R mRNA in the stalk/median eminence and anterior pituitary gland of follicular ewes subjected to short (3 h during one day) or prolonged (5 h daily during four consecutive days) footshock stimulation. To analyze relationship between expression of GnRH and GnRH-R genes with LH secretion the blood samples were collected at 10 min intervals to determine LH levels in control and stressed animals. The concentration of GnRH mRNA increased significantly in the preoptic area, anterior and ventromedial hypothalamus of ewes subjected to short stress. The prolonged stressful stimuli significantly decreased GnRH mRNA levels in all analyzed structures. In short stressed ewes the significant augmentation of mRNA encoding GnRH-R was detected in the preoptic area, entire hypothalamus, stalk/median eminence and anterior pituitary gland. The GnRH-R mRNA was significantly reduced in all tested structures of animals subjected to prolonged footshocking except for the preoptic area, where GnRH-R mRNA did not differ from control values. The changes in GnRH mRNA and GnRH-R mRNA levels under short or prolonged stress were associated with an increase or decrease of LH concentration in blood plasma, suggesting the existence of a direct relationship between GnRH mRNA and GnRH-R mRNA expression with LH secretion. The results indicate that the expression of both GnRH gene and GnRH-R gene, as well as LH secretion in ewes during the follicular phase of the estrous cycle, are dependent upon the kind of stress.

The effect of stress on the expression of GnRH and GnRH receptor genes in the discrete regions of the hypothalamus and pituitary of anestrous ewes.

Using the Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technique, the gonadotropin releasing-hormone (GnRH) mRNA and GnRH receptor (GnRH-R) mRNA were analyzed in the preoptic area (POA), anterior (AH) and ventromedial (VM) hypothalamus, stalk/median eminence (SME) and anterior pituitary gland (AP) of anestrous ewes subjected to short or prolonged footshock stimulation. No GnRH gene expression was detected in the SME and AP. The comparable levels of GnRH mRNA were found in the POA, AH and VM in control ewes. Short and prolonged footshock stimulation significantly increased GnRH mRNA in all analyzed tissue. The highest responses in GnRH mRNA to the short stress occurred in the POA whereas to the prolonged stress in the POA and VM. In non-stressed ewes the GnRH-R mRNA were detected in tissue continuum throughout the POA, AH, VM, SME and AP. The highest concentration of GnRH-R mRNA was detected in the SME. Short as well as prolonged stress stimuli caused an increase in GnRH-R mRNA levels in all analyzed tissue. The highest responses in GnRH-R mRNA expression were found in the VM. In spite of profound up-regulation of GnRH mRNA and GnRH-R mRNA under the short and prolonged stress conditions, the increase of luteinizing hormone (LH) secretion was noted only during acute stress. It is suggested that the increase of expression of GnRH and GnRH-R genes in anestrous ewes are not directly related to GnRH level and GnRH-R activity.

The role of GABA(A) and GABA(B) receptors in the control of GnRH release in anestrous ewes.

The paper reviews data concerning the involvement of GABA(A) and GABA(B) receptors in the control of GnRH secretion in anestrous ewes. Generally, GABA influences the GnRH release through GABA(A) and GABA(B) receptors located on perikaria of the GnRH neurons in the preoptic area (MPOA) or through the influence on beta-endorphinergic and catecholaminergic systems activity in MPOA and in ventromedial-infundibular region of the hypothalamus (VEN/NI). Stimulation of GABA(A) receptors in VEN/NI and MPOA attenuates GnRH release, while activation of GABA(B) receptors in MPOA decreases GnRH secretion, and in VEN/NI increases concentration of GnRH. The different neural mechanisms could be involved in this process: direct ligand action on the GABA(A) and GABA(B) receptors located on GnRH cells and axon terminals or indirect effect through the changes in the beta-endorphinergic and catecholaminergic systems activity in these structures of the brain.