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1

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

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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.
2

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

100%
Ciechanowska M., Lapot M., Paruszewska E., Brytan M., Przekop F.
Acta Neurobiologiae Experimentalis
|
2017
|
tom 77
|
nr Suppl.1
3

The central effect of Beta-endorphin and naloxone on kisspeptin and reamide-related peptide-3-the hypothalamic networks signalling gonadotropin-releasing hormone neurons in sheep

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Paruszewska E., Ciechanowska M., Laput M., Matusiak K., Nawrocka M., Przekop F.
Acta Neurobiologiae Experimentalis
|
2015
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tom 75
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nr Supl.
BACKGROUND AND AIMS: The aim of the present study was to investigate the effects of prolonged, intermittent infusion of β-endorphin or naloxone into the third cerebral ventricle of follicular phase ewes on kisspeptin (kiss 1) mRNA and RFamide-related peptide-3 (RFRP-3) mRNA levels in the hypothalamus. It was also examined the influence of β-endorphinergic stimulation or blockade on the expression of gonadotropin releasing hormone (GnRH) and GnRH receptor (GnRHR) proteins in the hypothalamic-pituitary unit and on luteinizing hormone (LH) secretion from the anterior pituitary gland. METHODS: The levels of GnRH and GnRHR proteins were analyzed using an enzyme-linked immunoabsorbent assay (ELISA) in selected tissue of the preoptic area-hypothalamic region: preoptic area (POA), anterior hypothalamus (AH), ventromedial hypothalamus (VM), stalk/median eminence (SME), and GnRHR in the anterior pituitary gland (AP). The Real-time PCR with SYBR Green dye was used for evaluation of kiss 1 mRNA in the POA and arcuate nucleus (ARC) and RFRP3 mRNA in the paraventricular nucleus (PVN) and in the dorsomedial hypothalamus (DMH). RESULTS: Stimulation of β-endorphin receptors significantly decreased the levels of GnRH protein and kiss 1 transcript in all analyzed structures and usually led to similar responses in the expression of GnRHR. Precisely, β-endorphin decreased the level of GnRHR protein in the POA, MBH, SME and AP, but had no significant influence on the receptor quantity in the AH. In addition, β-endorphin decreased LH secretion. Naloxone had an opposite effect on proteins biosynthetic level. CONCLUSIONS: The obtained results suggest that β-endorphin can modulate the biosynthesis and release of GnRH through complex changes in the expression of kiss 1 mRNA and GnRHR protein in the hypothalamus. It also appears, that in sheep β-endorphin influences GnRH/LH secretion by mechanism(s) excluding RFRP-3 neuronal system.
4

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

86%
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.
5

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

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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.
6

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

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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”.
7

Prolonged SOCS3 gene response following soman intoxication in the rat brain

86%
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”.
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