The differential effects of blocking retinal orexin receptors on the expression of retinal c-fos and hypothalamic Vip, PACAP, Bmal1, and c-fos in Male Wistar Rats.

Orexin A and B (OXA and OXB) and their receptors are expressed in the majority of retinal neurons in humans, rats, and mice. Orexins modulate signal transmission between the different layers of the retina. The suprachiasmatic nucleus (SCN) and the retina are central and peripheral components of the body's biological clocks; respectively. The SCN receives photic information from the retina through the retinohypothalamic tract (RHT) to synchronize bodily functions with environmental changes. In present study, we aimed to investigate the impact of inhibiting retinal orexin receptors on the expression of retinal Bmal1 and c-fos, as well as hypothalamic c-fos, Bmal1, Vip, and PACAP at four different time-points (Zeitgeber time; ZT 3, 6, 11, and ZT-0). The intravitreal injection (IVI) of OX1R antagonist (SB-334867) and OX2R antagonist (JNJ-10397049) significantly up-regulated c-fos expression in the retina. Additionally, compared to the control group, the combined injection of SB-334867 and JNJ-10397049 showed a greater increase in retinal expression of this gene. Moreover, the expression of hypothalamic Vip and PACAP was significantly up-regulated in both the SB-334867 and JNJ-10397049 groups. In contrast, the expression of Bmal1 was down-regulated. Furthermore, the expression of hypothalamic c-fos was down-regulated in all groups treated with SB-334867 and JNJ-10397049. Additionally, the study demonstrated that blocking these receptors in the retina resulted in alterations in circadian rhythm parameters such as mesor, amplitude, and acrophase. Finally, it affected the phase of gene expression rhythms in both the retina and hypothalamus, as identified through cosinor analysis and the zero-amplitude test. This study represents the initial exploration of how retinal orexin receptors influence expression of rhythmic genes in the retina and hypothalamus. These findings could provide new insights into how the retina regulates the circadian rhythm in both regions and illuminate the role of the orexinergic system expression within the retina.

Orexin antagonism and substance-P: Effects and interactions on polycystic ovary syndrome in the wistar rats.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder without definitive treatments. Orexin and Substance-P (SP) neuropeptides can affect the ovarian steroidogenesis. Moreover, there are limited studies about the role of these neuropeptides in PCOS. We aimed here to clarify the effects of orexins and SP in PCOS as well as any possible interactions between them. METHODS: For this purpose, the animals (n = five rats per group) received intraperitoneally a single dose of SB-334,867-A (orexin-1 receptor antagonist; OX1Ra), JNJ-10,397,049 (orexin-2 receptor antagonist; OX2Ra), and CP-96,345 (neurokinin-1 receptor antagonist; NK1Ra), alone or in combination with each other after two months of PCOS induction. The blocking of orexin and SP receptors was studied in terms of ovarian histology, hormonal changes, and gene expression of ovarian steroidogenic enzymes. RESULTS: The antagonists' treatment did not significantly affect the formation of ovarian cysts. In the PCOS groups, the co-administration of OX1Ra and OX2Ra as well as their simultaneous injections with NK1Ra significantly reversed testosterone levels and Cyp19a1 gene expression when compared to the PCOS control group. There were no significant interactions between the PCOS groups that received NK1Ra together with one or both OX1R- and OX2R-antagonists. CONCLUSION: The blocking of the orexin receptors modulates abnormal ovarian steroidogenesis in the PCOS model of rats. This suggests that the binding of orexin-A and -B to their receptors reduces Cyp19a1 gene expression while increasing testosterone levels.

Inhibition of the retinal orexin receptors affects the hypothalamic-pituitary-gonadal axis through retinal pituitary adenylate cyclase activating polypeptide (PACAP) in male Wistar rats.

Orexins A and B (OXA and OXB) and their receptors are expressed in the retina of both human and rodents and play a vital role in regulating signal transmission circuits in the retina. There is an anatomical-physiological relationship between the retinal ganglion cells and suprachiasmatic nucleus (SCN) through glutamate as a neurotransmitter and retinal pituitary adenylate cyclase-activating polypeptide (PACAP) as a co-transmitter. SCN is the main brain center for regulating the circadian rhythm, which governs the reproductive axis. The impact of retinal orexin receptors on the hypothalamic-pituitary-gonadal axis has not been investigated. Retinal OX1R or/and OX2R in adult male rats by 3 µl of SB-334867 (1 µg) or/and 3 µl of JNJ-10397049 (2 µg) were antagonized via intravitreal injection (IVI). Four time-periods were considered (3, 6, 12, and 24 h) for the controls without any treatment, SB-334867, JNJ-10397049, and SB-334867 + JNJ-10397049 groups. Antagonizing retinal OX1R or/and OX2R resulted in a significant elevation of retinal PACAP expression compared to control animals. In addition, expression of GnRH increased non-significantly in the hypothalamus over the 6 h of the study, and the serum concentration of LH decreased significantly in the SB-334867 group after 3 h of injection. Furthermore, testosterone serum levels declined significantly, especially within 3 h of injection; serum levels of progesterone were also exposed to a significant rise at least within 3 h of injection. However, the retinal PACAP expression changes were mediated by OX1R more effectively than by OX2R. In this study, we report the retinal orexins and their receptors as light-independent factors by which the retina affects the hypothalamic-pituitary-gonadal axis.

Interplay between polycystic ovary syndrome and hypothyroidism on serum testosterone, oxidative stress and StAR gene expression in female rats.

INTRODUCTION: Endocrine disorders such as polycystic ovary syndrome (PCOS) and hypothyroidism can cause infertility. There are evidence that they happen jointly in some circumstances. It still remains unknown, how these two illnesses interact and influence the body. METHODS: Accordingly, a five-group was designed, first is the control group, followed by the PCOS group. Estradiol valerate (EV) induced PCOS, the second group had only PCOS and the third, fourth and fifth groups were given varied dosages of propylthiouracil (PTU) to cause hypothyroidism after induction of PCOS. Steroidogenic acute regulatory protein (StAR) expression was measured in the ovaries, and serum was obtained to determine testosterone levels, as well as superoxide dismutase (SOD) as an antioxidant and malondialdehyde (MDA) as an oxidant. RESULTS: Based on radioimmunoassay data, testosterone levels were significantly higher in the PCOS group than the control group, and significantly lower (p Ë‚ .05) in PTU groups comparing with the PCOS group. According to the quantitative real-time polymerase chain reaction (qRT-PCR) data, the same results were obtained for the StAR gene as well. The data also indicated a positive correlation between these two. Although both oxidant and antioxidant level increased in PCOS group compared than control group, after hypothyroidism, oxidant level increased significantly (p Ë‚ .05), meanwhile antioxidant level decreased significantly (p Ë‚ .05). CONCLUSIONS: The results of this study illustrate that the presence of both PCOS and hypothyroidism alters the situation more than just PCOS. They also indicate that this situation is associated with imbalanced oxidative/antioxidative status.

Changes of plasma concentration and gene expression of ghrelin and leptin in rats receiving kisspeptin and morphine.

Kisspeptin is a hypothalamic peptide which stimulates hypothalamus- pituitary- gonadal (HPG) axis. Morphine is an alkaloid which suppresses reproduction. Ghrelin and leptin are metabolic peptides which play role in relaying information to the HPG axis. In the present study, the interaction effects of kisspeptin and morphine were investigated on plasma and gene expression levels of leptin and ghrelin. Twenty adult male Wistar rats randomized in four groups were received injection of saline, kisspeptin (1.00 nmol), morphine (5.00 mg kg-1) or Kisspeptin + morphine. Rats were received kisspeptin and morphine via third cerebral ventricular and subcutaneous injection, respectively. Ten male rats in two groups were received intravenous injection of saline or kisspeptin (7.50 nmol). Blood samples, hypothalamic and adipose tissue samples were collected. Plasma and gene expression levels of ghrelin and leptin were measured using the methods of enzyme-linked immunosorbent assay and real time-PCR, respectively. Morphine significantly increased plasma concentration and hypothalamic mRNA levels of ghrelin compared to saline while kisspeptin significantly decreased them compared to saline. Morphine significantly decreased plasma and mRNA levels of leptin in adipose tissue compared to saline, however, kisspeptin did not increase plasma and mRNA levels of leptin in adipose tissue compared to saline. Kisspeptin significantly decreased the effects of morphine on plasma concentration and hypothalamic gene expression levels of ghrelin compared to morphine alone, however, it did not affect morphine influence on plasma and leptin gene expression levels compared to morphine alone. Kisspeptin and morphine might partly be involved in the regulation of reproductive activity via regulation the metabolic hormones synthesis.

Phytotherapy of polycystic ovary syndrome: A review.

Background: Polycystic ovary syndrome (PCOS) is a complex heterogeneous disease with various symptoms, which can affect females of reproductive age. Endocrine and metabolic abnormalities such as infertility, being overweight or obese, type 2 diabetes, hyperandrogenism and increased luteinizing hormone (LH) are common in women with PCOS. Objective: This review aimed to assess the efficacy of non-chemical and herbal substances for PCOS recovery. Materials and Methods: The keywords "non-chemical treatment", "herbal treatment", "polycystic ovary syndrome" and "PCOS" were used to search for articles in the electronic databases PubMed/MEDLINE, Web of Science, Scopus, and Reaxys, published from January 2009 to December 2019. Results: 34 relevant studies were found and were briefly described in this review. The most effective herbal treatments in animal models of PCOS were used to restore abnormality in serum sex steroid profile, LH: follicle stimulating hormone ratio, steroidogenic enzymes, cardiovascular parameters, lipid profile, and glucose and estrous cycles. In PCOS patients, positive effects on PCOS due to reductions in testosterone, estrogen, LH, LH: follicle stimulating hormone ratio, and insulin levels were observed. Conclusion: The results of this reviewrevealed the variability and efficacy of phytotherapy and non-chemical treatments associated with PCOS disease. These findings may help future studies on the etiology and treatment of this syndrome.

Reporting The Effects of Exposure to Monosodium Glutamate on The Regulatory Peptides of The Hypothalamic-Pituitary-Gonadal Axis.

Monosodium glutamate (MSG) is a flavour enhancer that is used as a food additive (E621) in many parts of the world, especially in East Asian countries. However, in recent studies, it has been used as a neurotoxin because MSG is reported to cause neural degeneration in the hypothalamic arcuate of neonatal animals. The results of several studies show the negative effects of MSG injections on different parts of the hypothalamic-pituitary-gonadal (HPG) axis, in addition to its ability to inhibit secretion many reproductive neuropeptides, neurotrophic factors, and hormones, all of which play vital roles in the regulation of reproductive function. Oral administration or injection of large quantities of MSG into newborn animals results in a decrease in or overabundance of the production of many regulatory peptides of the male and female reproductive systems. In this review, we summarize the results of the most important studies that have examined the effect of oral consumption or injection of MSG on regulatory peptides of the HPG axis.

Ghrelin Is Effective on Passive Avoidance Memory by Altering the Expression of NMDAR and HTR1a Genes in the Hippocampus of Male Wistar Rats.

BACKGROUND: Memory-dependent psychological behaviors have an important role in life. Memory strengthening in adulthood to prevent its defects in aging is a significant issue. The ghrelin endogenous hormone improves memory by targeting glutamatergic and serotonergic circuits. Also, citicoline, a memory strengthening drug in aging, is not recommended to adults due to its side effects. The current study aims to test that ghrelin treatment, like citicoline, would improve passive avoidance memory via expression of the genes encoding the N-methyl-D-aspartate receptor (NMDAR1) and the serotonin receptor 1A (HTR1α) involved in this process. METHODS: Five groups of adult male rats received (1) saline (as control), (2) 0.5 mg/kg citicoline, or (3-5) 0.3, 1.5, and 3 nmol/µl ghrelin). The rats received the drugs via intra-hippocampal injection. Passive avoidance memory was determined using a shuttle box device. The latency to enter the dark chamber before (IL) and after (RL) injection and the total duration of the animal's presence in the light compartment (TLC) were evaluated. Then, the gene expression rates of NMDAR1 and HTR1α were measured by the Real-Time PCR. RESULTS: Ghrelin and citicoline had some similar and significant effects on passive avoidance memory, and both increased NMDAR1 and decreased HTR1α expression. CONCLUSION: Ghrelin, like citicoline, improves passive avoidance learning by altering the NMDAR1 and HTR1α expression in the hippocampus.

Rfamide-related peptide-3 suppresses the substance P-induced promotion of the reproductive performance in female rats modulating hypothalamic Kisspeptin expression.

RFamide-related peptide-3 (RFRP-3) has been postulated as the suppressor of the reproductive axis at hypothalamic, pituitary and gonadal levels. Considering the hypothalamic level, RFRP-3 can suppress the activity of gonadotropin-releasing hormone (GnRH) neurons and their upstream neuronal stimulator, namely; the kisspeptin neurons. The effects of the RFRP-3 on the other regulators of GnRH neurons, however, are not completely investigated. Furthermore, substance P (SP) has been known as one of the coordinators of GnRH/ luteinizing hormone (LH) and the kisspeptin/G protein-coupled receptor 54 (GPR54) systems. The present study was aimed at investigating the impacts of RFRP-3 on the effects of SP on the reproductive performance in ovariectomized female rats. After intracerebroventricular (ICV) cannulation, the rats were subjected to the ICV injection of either SP or RFRP-3 and simultaneous injection of them and their selective antagonists. Blood and hypothalamic samplings and also sexual behavioral test were carried out on two main groups of rats. The analyses of the results of LH radioimmunoassay, gene expression assay for hypothalamic Gnrh1, Kisspeptin and Gpr54 accompanied by sexual behavioral examination revealed that the SP administration promotes reproductive behavior and GnRH/LH system and upregulates Kisspeptin expression. The RFRP-3 administration suppressed reproductive behavior, GnRH / LH system and Kisspeptin expression; however, the simultaneous injection of SP and RFRP-3 was devoid of significant alterations in the assessed parameters. The results showed that RFRP-3 can modulates the impacts of SP on the reproductive performance in ovariectomized female rats in part through adjusting Kisspeptin expression.

Neuropeptide galanin and its effects on metabolic and reproductive disturbances in female rats with estradiol valerate (EV) - Induced polycystic ovary syndrome (PCOS).

A functional role of the neuropeptide galanin, executed through the three G-protein coupled receptor subtypes GAL1₋3, has been demonstrated in many biological systems and under pathological circumstances. Galanin is involved in many central and peripheral actions, in particular associated with endocrine mechanisms such as anterior pituitary hormone regulation, reproduction, glucose metabolism and also inflammation. The role of galanin in the pathology of the polycystic ovary syndrome (PCOS) and possible therapeutic effects are unknown. However, based on the well known neuroendocrine changes in PCOS patients, it may be assumed that galanin via effects on gonadotropin-releasing hormone (GnRH) secretory neurons could play a significant role in the development of PCOS. The aim of this study was to examine possible therapeutic effects of galanin on hormonal, metabolic and molecular parameters in PCOS. Accordingly, intraperitoneal injection of galanin in a dose- dependent manner in female PCOS rats induced a significant reduction in inflammatory markers (TNF-α, IL-6), an increase in FSH and a decrease in LH, insulin and testosterone (using ELISA kit) compared to the PCOS groups. Moreover, data from real-time quantitative PCR showed significantly ameliorated changes in the mRNA levels of the steroidogenic acute regulatory protein (StAR) and aromatase cytochrome P450 (CYP19). Taken together, galanin has neuroendocrine, anti- and pro-inflammatory and metabolic effects, and we therefore suggest that treatment with this peptide could represent new therapeutic approach for managing hormonal and metabolic disturbances in the PCOS disease.

Therapeutic potentials of the natural plant flavonoid apigenin in polycystic ovary syndrome in rat model: via modulation of pro-inflammatory cytokines and antioxidant activity.

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women of reproductive age, which is associated with increased androgens, chronic inflammation, and oxidative stress. Current research aims at determining the effect of flavonoid apigenin on the level of antioxidant and anti-inflammatory factors in ovarian tissue of rats with PCOS. In this study, 32 female Wistar rats were divided into four groups (n = 8): control, PCOS control, and treated groups (20 and 40 mg/kg apigenin). After 21 days of intervention, the serum levels of sex hormones and gonadotropins were measured. The ovarian tissue was removed for biochemical and histological studies. Research findings indicated that apigenin causes significant decrease in estrogen, testosterone levels, LH and LH to FSH ratio, and significant increase in progesterone and FSH levels in serum of treated groups compared to PCOS control group. Different doses of apigenin significantly decreased the inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and total oxidative status (TOS). The total antioxidant capacity (TAC) and superoxide dismutase (SOD) activity significantly increased in treated groups compared to the PCOS control group. The histological results indicated that the number of cysts and theca layer thickness significantly decreased and the number of corpora lutea and granulosa layer thickness significantly increased in the rats receiving the apigenin as compared to the PCOS control group. Flavonoid apigenin through antioxidant and anti-inflammatory properties can be used as an alternative method for treating patients with PCOS.

The effects of supraphysiological levels of testosterone on neural networks upstream of gonadotropin-releasing hormone neurons.

OBJECTIVES: Several pathological conditions are associated with hyper-production of testosterone; however, its impacts are not well understood. Hence, we evaluated the effects of supraphysiological levels of testosterone on gonadotropin-releasing hormone (GnRH) system in the hypothalamus of male rats. Also, we assessed the expression of two excitatory (kisspeptin and neurokinin-B) and two inhibitory (dynorphin and RFamide-related-peptide) neuropeptides upstream of GnRH neurons as possible routes to relay androgen information. MATERIALS AND METHODS: Gonadectomized (GDX) male rats received single injection of 100, 250 or 500 mg/kg testosterone undecanoate and three weeks later, posterior (PH) and anterior (AH) hypothalamus was dissected for evaluation of target genes using quantitative RT-PCR. RESULTS: We found that GnRH mRNA in the PH was high in GDX rats and 500 mg/kg testosterone reduced GnRH level expression. Finding revealed extremely high level of Kiss1 mRNA in the PH of GDX rats. However, in GDX rats treated with different levels of testosterone, Kiss1 expression was not significantly different than control. We also found that testosterone replacement increased the Kiss1 mRNA level in the AH. Moreover, neurokinin-B mRNA level in PH of GDX rats was similar to control. However, excess testosterone levels were effective in significantly inducing the down-regulation of neurokinin-B expression. The basal level of dynorphin mRNA was increased following testosterone treatments in the AH, where we found no significant difference in the level of RFamide-related-peptide mRNA between the experimental groups. CONCLUSION: Excess levels of testosterone could act differently from its physiological concentration to regulate hypothalamic androgen sensitive neurons to control GnRH cell.

Genome-Wide Distribution of Nascent Transcripts in Sperm DNA, Products of a Late Wave of General Transcription.

Mature spermatozoa contain a whole repertoire of the various classes of cellular RNAs, both coding and non-coding. It was hypothesized that after fertilization they might impact development, a claim supported by experimental evidence in various systems. Despite the current increasing interest in the transgenerational maintenance of epigenetic traits and their possible determination by RNAs, little remains known about conservation in sperm and across generations and the specificities and mechanisms involved in transgenerational maintenance. We identified two distinct fractions of RNAs in mature mouse sperm, one readily extracted in the aqueous phase of the classical TRIzol procedure and a distinct fraction hybridized with homologous DNA in DNA-RNA complexes recovered from the interface, purified after DNase hydrolysis and analyzed by RNA-seq methodology. This DNA-associated RNA (D RNA) was found to represent as much as half of the cell contents in differentiated sperm, in which a major part of the cytoplasmic material has been discarded. Stable complexes were purified free of proteins and identified as hybrids (R-loops) on the basis of their sensitivity to RNase H hydrolysis. Further analysis by RNA-seq identified transcripts from all the coding and non-coding regions of the genome, thus revealing an extensive wave of transcription, prior to or concomitant with the terminal compaction of the chromatin.

One-month of high-intensity exercise did not change the food intake and the hypothalamic arcuate nucleus proopiomelanocortin and neuropeptide Y expression levels in male Wistar rats.

OBJECTIVE: The hypothalamic arcuate nucleus proopiomelanocortin (POMC) and neuropeptide Y (NPY) circuitries are involved in the inhibition and stimulation of the appetite, respectively. The aim of this study was to investigate the effects of one-month lasting high-intensity exercise on the POMC mRNA and NPY mRNA expression in the above-mentioned brain structure and appetite and food intake levels. METHODS: Fourteen male Wistar rats (250±50 g) were used and kept in the well-controlled conditions (22±2 °C, 50±5% humidity, and 12 h dark/light cycle) with food and water ad libitum. The rats were divided into two groups (n=7): 1) control group (C, these rats served as controls) and 2) exercised group (RIE, these rats performed a high-intensity exercise for one month (5 days per week) 40 min daily with speed 35 m/min. The total exercise time was 60 min. The body weight and food intake were recorded continuously during the experiments. RESULTS: The results showed relative mRNA expression of POMC and NPY estimated in the hypothalamic arcuate nucleus. There were no significant differences in the NPY and POMC mRNAs expression levels and food intake between C and RIE groups. CONCLUSIONS: The present data indicate that one-month regular intensive exercise did not alter the levels of NPY and POMC mRNAs expression (as two important factors in the regulation of appetite) in the hypothalamic arcuate nucleus and food intake suggesting that this type of exercise itself is not an appropriate procedure for the body weight reduction.

The Impact of Morphine on Reproductive Activity in Male Rats Is Regulated by Rf-Amid-Related Peptide-3 and Substance P Adjusting Hypothalamic Kisspeptin Expression.

Obviously, opiates (e.g., morphine) are associated with the suppression and dysfunction of reproductive axis. It has been reported that substance P (SP) and RF-amid-related peptide-3 (RFRP-3) can exhibit anti-opioid effects in some regions of the nervous system. Moreover, SP and RFRP-3 are deemed as neuropeptides which exert modulatory and regulatory impacts on the function of the reproductive axis. The precise interactions of morphine with SP or RFRP-3 on the parameters of the reproductive activity, however, are not fully known. The present study was aimed to determine the impacts of the interaction of morphine either with SP or RFRP-3 on the hormonal and behavioral parameters of reproductive activity in male rats. In addition, it was aimed at determining whether the effects of these interactions rely on kisspeptin/G protein coupled receptor 54 (GPR54) pathway as the main upstream pulse generator and the mediator of the function of many inputs of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) system or not. Altogether, the resulted data from the sexual behavior tests, radioimmunoassay of LH/testosterone, and real-time quantitative PCR for the assessment of the expression of hypothalamic Kiss1, Gpr54, and Gnrh1 genes following concomitant administration of morphine with SP or RFRP-3 revealed that the suppressing effects of morphine on the parameters of reproductive axis activity can be affected by the administration of either RFRP-3 or SP. It is advocated that SP and RFRP-3, by the modulation of the expression of hypothalamic Kiss1, can possibly antagonize the effects of morphine on GnRH/LH system and sexual behavior.

Therapeutic effects of isoflavone-aglycone fraction from soybean (Glycine max L. Merrill) in rats with estradiol valerate-induced polycystic ovary syndrome as an inflammatory state.

Polycystic ovary syndrome (PCOS) is a proinflammatory/oxidative state resulting in metabolic dysregulation and ovarian dysfunction. Isoflavones in soybean seed possess anti-inflammatory/antioxidant properties. So, in this study, the effects of soybean isoflavone-aglycones on tissue inflammation, oxidative status, and ovarian histology in an animal model of PCOS were considered to achieve a novel therapeutic agent. Thirty-two female Wistar rats were divided into four groups (n = 8): a control group receiving the vehicle (CON-); a group with PCOS receiving no treatment (PCOS); and two groups of PCOS rats treated with soybean isoflavone-aglycone fraction (SISAF) at 50 and 100 mg/kg (SISAF50 and SISAF100). PCOS was induced by injecting a single dose of estradiol valerate (4 mg/kg, IM) dissolved in 0.2 ml of sesame oil following 60 days for the full development of polycystic ovaries. The SISAF treatments were administered orally once a day for 21 days. Then, the ovaries were harvested for the assessment of oxidative/antioxidative parameters, interleukin (IL)-6, and tumor necrosis factor (TNF)-α levels concomitant with histological studies. Treatment with SISAF reduced the number of cystic follicles and thickness of the theca layer, as well as increasing the number of corpora lutea and granulosa cells in PCOS rats. Also, SISAF treatment significantly decreased ovarian tissue IL-6 and TNF-α levels, and improved total oxidative/antioxidative status compared to the PCOS group. Isoflavones may provide therapeutic effects in PCOS owing to the antioxidant and anti-inflammatory properties.

Role of RF-amid Related Peptide-3 (RFRP-3) in Inhibitory Effect of Orexin A on Reproductive Function in the Animal Model of Male Wistar Rats.

AIMS: The aim of the present study is to examine the orexin A (OXA) signaling can leave any impact on the hypothalamic-pituitary-gonadal (HPG) axis and this impact can be relayed through the pathway of RF amide-related peptide-3 (RFRP-3, the mammalian ortholog of the avian gonadotropin-inhibitory hormone)/G-protein coupled receptor (GPR)-147 (RFRP-3 receptor) as a novel target for controlling of HPG axis in the male rats. MATERIALS AND METHODS: Male rats were categorized randomly into experimental groups including control vehicle, OXA, and its antagonists' group and went through to surgical cannulation into the third ventricle. After the intracerebroventricular injection of each solution, blood samples were collected for measurements of the LH and testosterone using radioimmunoassay method. Hypothalamus of the animals were isolated for analysis of the relative expression of Rfrp-3/Gpr-147 along with Gnrh gene by Real time-PCR. Also, in the different cohort of animal sexual behavior test was done. RESULTS: It was shown that OXA significantly decreases the mean serum level of the LH and testosterone and, at the same time, its antagonists neutralize this impact. Moreover, we demonstrated that OXA has reduced the hypothalamic gene expression of Gnrh and increased the expression of Rfrp-3 and Gpr-147 genes. While OXA antagonists neutralize this impact. CONCLUSIONS: The results of this study are related to the impact of orexin on the HPG axis. It is recommended that RFRP-3/GPR-147 system as the interneural pathway relay the data of orexin to the neurons of GnRH.

Morphine and kisspeptin influences on 5-α reductase and aromatase gene expression in adult male rats.

OBJECTIVES: Kisspeptin and opioids are important factors for controlling GnRH/LH secretion. In present study, influences of kisspeptin or morphine were investigated on 5α- reductase or aromatase (CYP19) genes expression in the hypothalamus, testis and liver. It aimed to investigate whether kisspeptin pathway may control morphine effects on plasma concentration of testosterone. MATERIALS AND METHODS: Twenty adult male rats in four groups received saline/saline, kisspeptin (1 nmol)/saline, morphine (5 mg/kg)/saline or kisspeptin/morphine respectively. Mean relative 5α-reductase and aromatase mRNA levels were determined by RT-PCR. RESULTS: Morphine/saline injection increased significantly mean relative mRNA levels of hypothalamic 5α-reductase or aromatase compared to saline/saline. While morphine/saline did not alter mRNA levels of them compared to saline/saline group in the testis and liver. Kisspeptin/saline did not significantly decrease mean relative 5α-reductase or aromatase genes expression compared to saline/saline group in the hypothalamus, testis and liver. Injections of kisspeptin/morphine did not significantly decrease mean relative 5α-reductase or aromatase genes expression compared to morphine/saline group. CONCLUSION: Up-regulation of hypothalamic 5α-reductase or aromatase mRNA levels may partly induce the inhibitory effects of morphine on GnRH/LH release. Different effects of morphine on aromatase or 5α- reductase genes expression levels in the liver and testis compared to brain may be partly due to different sensitivity or functions of them to morphine used dose.

Central Orexin A Affects Reproductive Axis by Modulation of Hypothalamic Kisspeptin/Neurokinin B/Dynorphin Secreting Neurons in the Male Wistar Rats.

It is an established fact that orexin plays an important role in regulating the reproductive axis and the secretions of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH). However, its precise cellular and molecular mechanisms are not fully recognized. Accordingly, the aim of the present study is to find out whether the central injection of orexin A (OXA) and its antagonists, SB-334867 (as orexin receptor antagonist 1; OX1RA) and JNJ-10397049 (as orexin receptor antagonist 2; OX2RA), either alone or in combination, can leave any impact on the reproductive axis (either hormonal or behavioral) in the male Wistar rats. Furthermore, in order to see whether OXA signals can be relayed through the pathway of kisspeptin/neurokinin B/dynorphin (known as KNDy neurons, a neural network which works upstream of GnRH neurons) or not, the relative gene expression of these neuropeptides were measured. Overall, the data from radioimmunoassay revealed that OXA significantly decreases the mean serum level of LH and testosterone and, in a similar vein, its antagonists neutralize this impact. Moreover, data from real-time quantitative PCR indicated that OXA has significantly reduced the hypothalamic expression of Gnrh. In this line, the gene expressions of Kisspeptin and Neurokinin b decreased. However, OXA antagonists neutralize this impact. Also, the expression of Dynorphin gene was upregulated by the following application of the OXA. The results of this study are related to the impact of orexin on the reproductive axis. It is recommended that KNDy neurons as the interneural pathway relay the information of orexin to the GnRH neurons.

Various responses of male pituitary-gonadal axis to different intensities of long-term exercise: Role of expression of KNDYrelated genes.

The essential role of regular physical activity has been emphasized for maintaining a healthy life. However, unfortunately, during the last few decades, the lifestyle of people has led to a decrease in physical activity. Research studies have shown that exercise of different intensities is applied on reproductive performance indices, luteinizing hormone (LH) and testosterone (T), with different effects. Nevertheless, the molecular and cellular mechanisms underlying its function are not completely understood. Therefore, this study aimed to evaluate the role of kisspeptin, neurokinin-B and pro-dynorphin (KNDY) gene-expression changes located in the upstream of GnRH neurons in transferring the effects of different long-term exercise intensities on male reproductive axis. Twenty-one adult Wistar rats were randomly divided into control, 6-month regular moderate exercise (RME-6) and 6-month regular intensive exercise (RIE-6). In moderate and intensive exercise groups, rats were treated 5 days a week for 60 min, at 22 and 35 m/min, respectively. Finally, the hypothalamic arcuate nucleus was isolated and the relative gene expression of kisspeptin (Kiss1), neurokinin-B (Nkb), pro-dynorphin (Pdyn) and gonadotropin-releasing hormone (Gnrh) genes were measured by realtime polymerase chain reaction method. The results showed that RIE-6 treatment decreased Gnrh and increased Pdyn mRNA levels in the arcuate nucleus. Furthermore, although RME-6 treatment decreased Nkb and increased Pdyn mRNA levels, the Gnrh mRNAwas not affected. Regarding the Gnrh mRNA levels and serum concentrations of reproductive indices (LH and T), moderate exercise did not impose harmful effects on the hypothalamic-pituitary-gonadal axis than intensive exercise. The different impacts of diverse long-term exercise intensities on the male pituitary-gonadal axis maybe relay by the various changes in hypothalamic Nkb and Pdyn gene expressions.