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1

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

100%
Ciechanowska M., Lapot M., Mateusiak K., Przekop F.
Reproductive Biology
|
2010
|
tom 10
|
nr 2
2

Molecular mechanisms participating in inhibition of GnRH/LH secretion induced by CRH

88%
Ciechanowska M., Lapot M., Paruszewska E., Brytan M., Przekop F.
Acta Neurobiologiae Experimentalis
|
2017
|
tom 77
|
nr Suppl.1
INTRODUCTION: It is generally accepted that corticotropin-releasing hormone (CRH) is the central mediator of stress-activated changes in the pituitary-adrenal axis because it results in the release of adrenocorticotropic hormone (ACTH) and finally increases the levels of cortisol. In some situations CRH also inhibits the release of GnRH and it has been proposed as a mediator of the anti‑reproductive effects of stress. AIM(S): This study aimed to explain how prolonged activation or inhibition of CRH‑ergic activity affected molecular processes governing GnRH/LH secretion in follicular-phase sheep. METHOD(S): The study included two experimental approaches: first, we investigated the effect of CRH or CRH antagonist (α‑helical CRH 9‑41; CRH‑A) on GnRH and GnRH receptor (GnRHR) biosynthesis in the hypothalamus and on GnRHR in the anterior pituitary gland (AP) using an immunoassay (ELISA). This analysis was supplemented by radioimmunoassay (RIA) method for LH; second, we used Real-time PCR to analyse the influence of CRH and CRH‑A on the levels of kisspeptin (Kiss 1) mRNA in the preoptic area (POA) and ventromedial hypothalamus including arcuate nucleus (VMH/ARC). RESULTS: Our results show that stimulation or inhibition of CRH receptors significantly decreased or increased GnRH biosynthesis in the hypothalamus, respectively, and led to differentresponsesin the expression of GnRHR. CRH increased GnRHR abundance in the POA, but decreased it in the hypothalamus and in the AP. Blockade of CRH receptors had the opposite effect on the level of post‑translational product of GnRHR gene. In addition, administration of CRH decreased plasma LH concentration and Kiss1 mRNA in the POA and VMH/ARC, while CRH-A exerted an opposite action. CONCLUSIONS: The study demonstrates that CRH-ergic neurotransmission is involved in the regulatory pathways of GnRH and GnRHR biosynthesis in the hypothalamic-pituitary unit of follicular-phase sheep conceivably via mechanisms in which Kiss 1 participate. FINANCIAL SUPPORT: This work was supported by grant National Science Center Poland No UMO-2012/05/B/ NZ4/02443.
3

Implication of GABA-ergic system on GnRH and GnRHR biosynthesis and LH secretion in the hypothalamic-pituitary unit of follicular-phase ewes

88%
Lapot M., Brytan M., Kowalczyk M., Przekop F., Ciechanowska M.
Acta Neurobiologiae Experimentalis
|
2019
|
tom 79
|
nr Suppl.1
INTRODUCTION: An inverse relationship between GnRH transcript level and GABA neuronal activity has been suggested; in particular, that GABA at the hypothalamic level may exert a suppressive effect on subsequent steps of GnRH biosynthesis. This inhibitory action of GABA seems to be mediated mainly via a GABA type A receptor (GABAAR) mechanism. AIM(S): In the present study, we analysed the effects of GABAAR activation or inhibition on the levels of post-translational products of the genes encoding the GnRH ligand and GnRH receptor (GnRHR) in the hypothalamic-pituitary unit of follicular-phase ewes using an in vivo infusion model. METHOD(S): Prolonged intermittent infusions of small doses of the GABAAR agonist, muscimol, or an antagonist, bicuculline, were performed into the third cerebral ventricle of ewes. Enzyme‑linked immunosorbent assay (ELISA) was used to investigate the effects of drugs on GnRH and GnRHR levels in the preoptic area (POA), anterior (AH) and ventromedial (VMH) hypothalamus, stalk/median eminence (SME), and GnRHR in the anterior pituitary gland (AP). The radioimmunoassay (RIA) method was also chosen for determining the level of LH in blood plasma. RESULTS: The study showed that activation or blockade of GABAAR significantly decreased or increased, respectively, GnRH concentration in all analysed structures and led to analogous changes in plasma LH concentration. Similar muscimol‑ and bicuculline‑related alterations were observed in the levels of GnRHR. CONCLUSIONS: On the basis of these results, it is suggested that the GABAergic system in the hypothalamus of follicular-phase sheep affects GnRH and GnRHR biosynthesis either directly or via GABAergic-sensitive interneurons, leading to suppression in the secretory activity of the hypothalamic‑pituitary GnRH/LH system.
4

The role of cortisol and kisspeptin in suppression of the GnRH/LH secretion in follicular-phase ewes subjected to prolonged stress

88%
Ciechanowska M., Lapot M., Kowalczyk M., Brytan M., Przekop F.
Acta Neurobiologiae Experimentalis
|
2019
|
tom 79
|
nr Suppl.1
INTRODUCTION: A contrary relation between prolonged stress and normal reproductive efficiency has frequently been observed in domestic animals, and the action of various stressors is manifested primarily by an increase in the level of glucocorticoids in blood. AIM(S): This study aimed to determine the central and peripheral mechanisms governing GnRH biosynthesis and LH secretion in prolonged-stressed follicular‑phase sheep. They included two main experimental approaches: (1) an investigation of the effects of physical stress on GnRH and GnRH receptor (GnRHR) biosynthesis, and (2) examination of the influence of stressor on levels of mRNAs encoding kisspeptin (Kiss1) and the Kiss1 receptor (Kiss1r). Furthermore, plasma LH and cortisol concentrations were also measured. METHOD(S): The ELISA technique was used to analyse the effects of stress on levels of post-translational products of genes encoding the 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 effects of stress on Kiss1 mRNA levels in the POA and the VMH the including arcuate nucleus (VMH/ARC), and on Kiss1r mRNA abundance in POA‑hypothalamic structures. These analyses were supplemented by radioimmunoassay (RIA) and ELISA methods for the measurement of LH and cortisol levels in blood, respectively. RESULTS: Stress decreased GnRH and GnRHR biosynthesis in the hypothalamus, and GnRHR in the AP. Moreover, stress lowered plasma LH concentration and levels of Kiss1 mRNA in the POA and VMH/ARC as well as Kiss1r mRNA in these structures and in the SME. An increase in plasma cortisol concentration under stress conditions was also observed. CONCLUSIONS: This study demonstrates that stress affects GnRH/GnRHR biosynthesis and LH secretion in follicular-phase sheep, conceivably via both central and peripheral mechanisms including Kiss1 neuronal activity and action of cortisol.
5

Molecular mechanisms participating in inhibition of GnRH/LH secretion induced by CRH

88%
Ciechanowska M., Lapot M., Paruszewska E., Brytan M., Przekop F.
Acta Neurobiologiae Experimentalis
|
2017
|
tom 77
|
nr Suppl.1
6

Changes in GnRH and GnRH receptor biosynthesis in the hypothalamic-pituitary unit of anestrous ewes after infusion of GnRH into the third cerebral ventricle

76%
Lapot M., Przekop F., Paruszewska E., Antkowiak B., Malewski T., Ciechanowska M.
Acta Neurobiologiae Experimentalis
|
2017
|
tom 77
|
nr Suppl.1
INTRODUCTION: Physiological regulation of GnRH secretion in mammals is associated with complex interplay between excitatory and inhibitory neurotransmitter and neurohormone systems within the hypothalamus that mediate oestrogen signals to GnRH cells. Among numerous studies on the neuroendocrine processes controlling GnRH secretion only a few research have revealed that hypothalamic GnRH acting locally may also participate in the regulation of its own release, but the mechanisms involved remain poorly understood. AIM(S): This study was designed to evaluate the effect of prolonged intermittent infusion of small doses of exogenous GnRH on GnRH and GnRH receptor (GnRHR) biosynthesis in the hypothalamus, GnRHR expression in the anterior pituitary gland (AP), and on LH secretion in sexually inactive sheep. METHOD(S): Studies were conducted on 3–4-year-old Polish Merino ewes during the middle of the anestrous season. Infusions of exogenous GnRH (0.2 µg GnRH per animal daily) were performed into the third cerebral ventricle for 3 consecutive days. The control for GnRH-treated group was animals infused with equivalent to GnRH volume of Ringer’s solution. The measurement of GnRH and GnRHR levels was performed using an enzyme-linked immunosorbent assay (ELISA). Plasma LH concentration was analysed by a double-antibody radioimmunoassay (RIA). RESULTS: The results of this study demonstrate that prolonged infusion of small doses of GnRH into the third cerebral ventricle of anestrous ewes increased drastically GnRH and GnRHR levels in the hypothalamus, but decreased GnRHR expression in the AP and also reduced LH secretion. CONCLUSIONS: The study indicates the existence of ultrashort loop feedback mechanism of GnRH release from the hypothalamus in which hypothalamic GnRHR participate. Decreased expression of GnRH in the AP and diminished LH secretion in GnRH-treated ewes provide indirect evidence for suppressive effect of exogenous GnRH on GnRH release from hypothalamic nerve terminals. FINANCIAL SUPPORT: This work was supported by grant National Science Center Poland No UMO-2012/05/B/ NZ4/02443.
7

The influence of dapaminergic system inhibition on kisspeptin and rfamide-related peptide-3 transcripts and gonadotropin releasing-hormone/receptor proteins in anestrous ewes

76%
Lapot M., Ciechanowska M., Kowalczyk M., Mateusiak K., Paluch M., Antkowiak B., Przekop F.
Acta Neurobiologiae Experimentalis
|
2015
|
tom 75
|
nr Supl.
BACKGROUND AND AIMS: The aim of this study was to explain how prolonged inhibition of central dopaminergic activity affected the neuroendocrine processes controlling GnRH/gonadotropins secretion in the hypothalamus and in the anterior pituitary gland compartments of sheep during the non-breeding time. In this purpose, we investigated the effects of prolonged, intermittent infusions of small doses of sulpiride (dopaminergic D2 receptors antagonist) on transcript levels of the two central regulators of the hypothalamic-pituitary reproductive system – kiss 1 and RFRP-3. It was also examined the influence of sulpiride on the GnRH and GnRH receptor (GnRHR) biosynthesis in the hypothalamus-pituitary unit. Additionally, these analyses were complemented by the study of plasma LH concentration. METHODS: The levels of kiss 1 and RFRP-3 transcripts and GnRH and GnRHR proteins were analyzed using Real-time PCR technique or an enzyme-linked immunoabsorbent assay (ELISA), respectively. Plasma LH concentration was measured by a doubleantibody radioimmunoassay. RESULTS: Pharmacological blockade of D2 receptors by sulpiride significantly increased kiss 1 mRNA levels in the preoptic area and in the arcuate nucleus/ventromedial hypothalamus and decreased the RFRP-3 mRNA in the paraventricular nucleus. The abolition of dopaminergic neurons activity also resulted in augmentation of the GnRH and GnRHR protein levels in the entire hypothalamus and led to similar changes in the expression of GnRHR in the anterior pituitary gland. The increase of kiss 1 transcription and GnRH biosynthesis has appeared with a concomitant increase of LH secretion. CONCLUSIONS: Similar direction of changes of kiss 1 transcript and GnRH protein levels with LH secretion strongly suggest that the inhibition of D2 receptors has a stimulatory effect on kiss1/ GnRH biosynthesis and consequently on LH release. It is likely, that dopamine inhibits gonadotropin synthesis and release in anestrous period through complex actions of kiss1/RFRP-3 neurons on GnRH cells.
8

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

76%
Lapot M., Ciechanowska M., Malewski T., Misztal T., Mateusiak K., Przekop F.
Reproductive Biology
|
2007
|
tom 07
|
nr 1
9

Ekspresja genu GnRH i genu receptora GnRH (GnRHR) w układzie podwzgórzowo-przysadkowym owcy w różnych stanach fizjologicznych

76%
Ciechanowska M., Lapot M., Malewski T., Mateusiak K., Misztal T., Przekop F.
Postępy Nauk Rolniczych
|
2010
|
tom 62
|
nr 4
10

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

76%
Ciechanowska M., Lapot M., Antkowiak B., Mateusiak K., Paruszewska E., Paluch M., Przekop F.
Acta Neurobiologiae Experimentalis
|
2015
|
tom 75
|
nr Supl.
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.
11

The influence of soman on the expression of tp53 gene in the rat brain; distant effect

76%
Brytan M., Kowalczyk M., Lapot M., Paruszewska E., Antkowiak B., Przekop F., Ciechanowska M.
Acta Neurobiologiae Experimentalis
|
2017
|
tom 77
|
nr Suppl.1
INTRODUCTION: The organophosphorus compound soman (GD), an irreversible inhibitor of cholinesterases, produces seizure activity and related brain damage. Studies using various biochemical markers of programmed cell death indicate apoptotic rather than necrotic mechanism of GD-induced acute cell damage in the brain. One of the most important links between the proliferation and cell death machinery is the tumor suppressor p53, which as a guardian of the genome and the element promoting apoptosis makes it a prime target for a prognostic factor. AIM(S): The aim of this study was to examine distant effects of poisoning with a small, repeated dose of GD on the expression of mRNA encoding p53 protein in the rat brain. METHOD(S): The study was performed on maternal generation (F0) and on first filial generation (F1) of Wistar rats. Low clinically asymptomatic dose of GD (0.2×LD50) was administered by subcutaneous repeated injections, first in pregnancy and subsequently during the lactation period. Six months after the end of poisoning the animals were euthanised and brain structures (hippocampus, cerebellum and piriform cortex) were isolated aseptically for evaluation of p53 mRNA. To determine p53 transcript levels Real-Time PCR with SYBR Green dye was applied. RESULTS: GD action resulted in a significant increase of p53 transcript in the cerebellum and in the piriform cortex of both F0 and F1 females as well as in F1 males. The significant elevation of p53 mRNA level in the hippocampus was observed only in F1 females. CONCLUSIONS: The study demonstrates that GD causes distant changes in the expression of p53 mRNA in the rat brain. Increased expression of p53 mRNA provides indirect evidence that GD-induced distant disorders may include DNA damage and cell cycle disturbances leading to cell dysfunction and their elimination via apoptosis. FINANCIAL SUPPORT: This work was supported by Polish Ministry of Science and Higher Education No O R00 0042 08, “Soldier as a precise weapon – individual sets and kits”.
12

Prolonged SOCS3 gene response following soman intoxication in the rat brain

76%
Paruszewska E., Lapot M., Brytan M., Antkowiak B., Kowalczyk M., Ciechanowska M.
Acta Neurobiologiae Experimentalis
|
2017
|
tom 77
|
nr Suppl.1
INTRODUCTION: In comparison with well documented data concerning the mechanisms of acute neurotoxic action of nerve agents such as soman (GD), the immunomodulatory properties of these compounds are still poorly understood, especially considering their long‑term effects. One promising candidate for mediation of GD-induced immunomodulation seems to be a suppressor of cytokine signaling 3 (SOCS 3) – an intracellular protein which exhibits a wide variety of physiological effects on immune cell function. There also exists strong evidence to support SOCS3 as a crucial regulator of many disease processes in the central nervous system. AIM(S): The aim of the present study was to determine whether perinatal exposure to GD exerted distant action on the expression of mRNA encoding SOCS3 in selected tissue of the rats brain. METHOD(S): Studies were conducted on maternal generation (F0) and on first filial generation (F1) of Wistar rats. F0 animals were treated subcutaneously with a low (0.2×LD50) repeated dose of soman (o-pinacolyl methylphosphonofluoridate). GD was administrated first, in pregnancy, and subsequently during the lactation period. Six months after termination of GD exposure animals were anesthetised and immediately hippocampus, cerebellum and piriform cortex were obtained for subsequent analysis. Real-Time PCR with SYBR Green dye was used to evaluate the level of SOCS3 mRNA in selected structures of the brain. RESULTS: Intoxication with GD decreased significantly SOCS3 mRNA levels in the cerebellum and in the piriform cortex in F0 females and in their offspring of both sexes. The analogous tendency, but without statistical significance, was observed in the hippocampus of all experimental animals. CONCLUSIONS: The current data do not clarify distant signs and symptoms of soman exposure; however, a decrease in expression of SOCS3 following intoxication with GD may suggest a functional role of this protein in pathogenesis of GD-induced neurological disorders. FINANCIAL SUPPORT: This work was supported by Polish Ministry of Science and Higher Education No O R00 0042 08, “Soldier as a precise weapon – packages and sets”.
13

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

76%
Ciechanowska M., Lapot M., Malewski T., Misztal T., Mateusiak K., Przekop F.
Acta Neurobiologiae Experimentalis
|
2007
|
tom 67
|
nr 1
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.
14

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

76%
Lapot M., Ciechanowska M., Malewski T., Mateusiak K., Misztal T., Przekop F.
Reproductive Biology
|
2008
|
tom 08
|
nr 2
15
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Effects of corticotropin releasing hormone and corticotropin releasing hormone antagonist on biosynthesis of gonadotropin releasing hormone and gonadotropin releasing hormone receptor in the hypothalamic-pituitary unit of follicular-phase ewes and contribution of kisspeptin

64%
Ciechanowska M., Kowalczyk M., Lapot M., Malewski T., Antkowiak B., Brytan M., Winnicka I., Przekop F.
Journal of Physiology and Pharmacology
|
2018
|
tom 69
|
nr 3
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