ABSTRACT
BACKGROUND: Gonadotropin-releasing hormone (GnRH) receptor, a rhodopsin-like G-protein coupled receptor (GPCR) family member involved in GnRH signaling, is reported to be expressed in several tumors including glioblastoma multiforme (GBM), one of the most malignant and aggressive forms of primary brain tumors. However, the molecular targets associated with GnRH receptor are not well studied in GBM or in other cancers. The present study aims at investigating the effect of GnRH agonist (Gosarelin acetate) on cell proliferation and associated signaling pathways in GBM cell line, LN229. METHODS: LN229 cells were treated with different concentrations of GnRH agonist (10-10 M to 10-5 M) and the effect on cell proliferation was analyzed by cell count method. Further, total protein was extracted from control and GnRH agonist treated cells (with maximum reduction in cell proliferation) followed by trypsin digestion, labeling with iTRAQ reagents and LC-MS/MS analysis to identify differentially expressed proteins. Bioinformatic analysis was performed for annotation of proteins for the associated molecular function, altered pathways and network analysis using STRING database. RESULTS: The treatment with different concentrations of GnRH agonist showed a reduction in cell proliferation with a maximum reduction of 48.2% observed at 10-6 M. Quantitative proteomic analysis after GnRH agonist treatment (10-6 M) led to the identification of a total of 29 differentially expressed proteins with 1.3-fold change (23 upregulated, such as, kininogen-1 (KNG1), alpha-2-HS-glycoprotein (AHSG), alpha-fetoprotein (AFP), and 6 downregulated, such as integrator complex subunit 11 (CPSF3L), protein FRG1 (FRG1). Some of them are known [KNG1, AHSG, AFP] while others such as inter-alpha-trypsin inhibitor heavy chain H2 (ITIH2), ITIH4, and LIM domain-containing protein 1 (LIMD1) are novel to GnRH signaling pathway. Protein-protein interaction analysis showed a direct interaction of KNG1, a hub molecule, with GnRH, GnRH receptor, EGFR and other interactors including ITIH2, ITIH4 and AHSG. Overexpression of KNG1 after GnRH agonist treatment was validated using Western blot analysis, while a significant inhibition of EGFR was observed after GnRH agonist treatment. CONCLUSIONS: The study suggests a possible link of GnRH signaling with EGFR signaling pathways likely via KNG1. KNG1 inhibitors may be investigated independently or in combination with GnRH agonist for therapeutic applications.
Subject(s)
Brain Neoplasms/metabolism , Cell Proliferation/drug effects , Glioblastoma/metabolism , Gonadotropin-Releasing Hormone/biosynthesis , Receptors, LHRH/biosynthesis , Animals , Antineoplastic Agents, Hormonal/pharmacology , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Cell Line, Tumor , Chromatography, Liquid , Computational Biology , Glioblastoma/genetics , Glioblastoma/pathology , Gonadotropin-Releasing Hormone/agonists , Gonadotropin-Releasing Hormone/genetics , Goserelin/pharmacology , Humans , Proteomics/methods , Receptors, LHRH/genetics , Signal Transduction/drug effects , Tandem Mass SpectrometryABSTRACT
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.
Subject(s)
Estrous Cycle/metabolism , Gonadotropin-Releasing Hormone/biosynthesis , Hypothalamo-Hypophyseal System/drug effects , Kisspeptins/metabolism , Receptors, GABA-A/metabolism , Receptors, LHRH/biosynthesis , Animals , Bicuculline/pharmacology , Female , GABA-A Receptor Agonists/pharmacology , GABA-A Receptor Antagonists/pharmacology , Gonadotropin-Releasing Hormone/blood , Hypothalamo-Hypophyseal System/metabolism , Muscimol/pharmacology , Neurons/metabolism , SheepABSTRACT
AIM: To evaluate the effects of a treatment with leuprolide acetate (LA) on bladder overactivity as well as the expression of gonadotropin releasing hormone receptor (GnRH-R), and neurofilaments NF68 and NF200 in female rats with overactive bladder induced by castration. METHODS: Changes in the urodynamic parameters were determined in SHAM, ovariectomized (OVX) and ovariectomized rats treated with LA (OVX-LA). A semi-quantitative analysis for the expression pattern of GnRH-R and neurofilaments NF68 and NF200 were determined. RESULTS: Forty-three days after ovariectomy, rats from the OVX group have significant lower values for intercontractile interval (ICI) and compliance (C); as well as higher values for basal bladder pressure (BP) and frequency of non-voiding contractions (NVC). The systemic application of LA increased voiding volume (Vv) and pressure threshold (ThP) in the OVX-LA animals. The application of LA reduced the high frequency of NVC in the OVX rats. No significant differences were found for Vv and NVCs between the OVX-LA vs SHAM groups. At the mid part of the bladder, the presence of GnRH-R was evidenced in the urothelium of the SHAM group. The OVX animals showed different pattern of immunolabeling for GnRH-R as well as for neurofilaments NF200 and NF68, whereas in the OVX-LA group the immunofluorescence pattern was similar to the one seen in SHAM bladders (P < 0.05 for OVX vs OVX + LA). CONCLUSIONS: the results suggest that systemic application of LA can improve bladder dysfunction in castrated rats, and perhaps considered as a treatment for overactive bladder conditions secondary to menopause.
Subject(s)
Leuprolide/pharmacology , Ovariectomy , Receptors, LHRH/agonists , Urodynamics/drug effects , Animals , Compliance/drug effects , Female , Muscle Contraction/drug effects , Neurofilament Proteins/biosynthesis , Neurofilament Proteins/genetics , Rats , Rats, Wistar , Receptors, LHRH/biosynthesis , Urinary Bladder/drug effects , Urinary Bladder/metabolism , Urothelium/drug effects , Urothelium/metabolismABSTRACT
BACKGROUND: Insulin sensitizers like metformin and pioglitazone are clinically used since last decades for the treatment of PCOS, but their efficacy and possible role in PCOS patients remains questionable. Also, the mechanism by which these insulin sensitizers show effect is not clear. AIM OF THE STUDY: To evaluate the effect of metformin and pioglitazone on leutinizing hormone and follicle stimulating hormone receptor mRNA expression, hyperandrogenism and insulin resistance in high fat diet induced and letrozole induced PCOS in rats. METHODS: Pre-pubertal female rats were divided into four groups: group I received normal pellet diet and group II, III and IV received high fat diet. After 105 d of dietary manipulation, metformin and pioglitazone treatment was given to group III and group IV animals respectively for 21 d. Similarly, adult female rats were divided into four groups: group I received 1% carboxymethyl cellulose (CMC) and group II, III, IV received letrozole for 21 d. After 21 d of letrozole administration, metformin and pioglitazone treatment was given to group III and group IV animals respectively for 21 d. Oral glucose tolerance test, lipid profile, fasting glucose, insulin, estrus cycle, hormonal profile, ovary weight, leutinizing hormone receptor and follicle stimulating hormone receptor mRNA expression was measured. Polycystic ovarian morphology was assessed through histopathological changes of ovary. RESULTS AND CONCLUSION: Metformin and pioglitazone treatment improve both metabolic and reproductive parameters of PCOS including hyperinsulinemia and hyperandrogenism. LH receptor and FSH receptor mRNA expression were altered by pioglitazone and metformin treatment.
Subject(s)
Hyperandrogenism/genetics , Hypoglycemic Agents/pharmacology , Insulin Resistance/genetics , Metformin/pharmacology , Pioglitazone/pharmacology , Polycystic Ovary Syndrome/drug therapy , Receptors, LHRH/biosynthesis , Animals , Body Weight/drug effects , Carboxymethylcellulose Sodium/administration & dosage , Diet, High-Fat/adverse effects , Female , Glucose Tolerance Test , Humans , Insulin/metabolism , Letrozole/toxicity , Polycystic Ovary Syndrome/chemically induced , Polycystic Ovary Syndrome/pathology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Receptors, LHRH/geneticsABSTRACT
As an essential mediator in the Gonadotropin-releasing hormone (GnRH) signaling pathway, GnRH receptor (GnRHR) coupled to GnRH, plays an important role in activating the downstream pathway after stimulating a series of cascades to regulate reproduction. To detect the existence of GnRHR and potential GnRH signaling pathway, we cloned and characterized GnRHR in the Chinese mitten crab, Eriocheir sinensis (named EsGnRHR). The full-length EsGnRHR cDNA is 2038â¯bp in length, including an open reading frame (ORF) of 1566â¯bp, a 57â¯bp 5'-untranslated region (5'-UTR) and a 415â¯bp 3'-UTR. Prediction of transmembrane domains in protein sequence revealed that the EsGnRHR protein contained seven hydrophobic transmembrane regions (TMs). Reverse transcription PCR revealed that EsGnRHR was mainly expressed in the thoracic nerve group and ovary, and weakly distributed in the testis and brain. In situ hybridization further demonstrated that EsGnRHR mRNA was localized at the protocerebrum and deutocerebrum. In the ovary and testis, the hybridization signal was dominantly at the earlier developmental stages. The signal was mainly localized in the cytoplasm cell in the ovary, and in the epithelium cell in the testis. During the different stages of gonadal development, EsGnRHR displayed increasing trends in both female and male when analyzed by quantitative real-time PCR, suggesting that EsGnRHR was involved in controlling gonadal development. Our study provides important information for further research on the molecular mechanisms underlying crab development.
Subject(s)
Arthropod Proteins , Brachyura , Cloning, Molecular , Gene Expression Regulation/physiology , Receptors, LHRH , Animals , Arthropod Proteins/biosynthesis , Arthropod Proteins/genetics , Brachyura/genetics , Brachyura/metabolism , Female , Male , Ovary/metabolism , Receptors, LHRH/biosynthesis , Receptors, LHRH/genetics , Testis/metabolismABSTRACT
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.
Subject(s)
Anestrus/metabolism , Dopamine D2 Receptor Antagonists/pharmacology , Gonadotropin-Releasing Hormone/biosynthesis , Kisspeptins/metabolism , Neuropeptides/metabolism , Receptors, LHRH/biosynthesis , Sulpiride/pharmacology , Animals , Female , Hypothalamus/drug effects , Hypothalamus/metabolism , Luteinizing Hormone/blood , Pituitary Gland, Anterior/drug effects , Pituitary Gland, Anterior/metabolism , SheepABSTRACT
Swine are the only livestock species that produce both the second mammalian isoform of gonadotropin-releasing hormone (GNRH2) and its receptor (GNRHR2). Previously, we reported that GNRH2 and GNRHR2 mediate LH-independent testosterone secretion from porcine testes. To further explore this ligand-receptor complex, a pig model with reduced GNRHR2 expression was developed. Small hairpin RNA sequences targeting porcine GNRHR2 were subcloned into a lentiviral-based vector, lentiviral particles were generated and microinjected into the perivitelline space of zygotes, and embryos were transferred into a recipient. One GNRHR2 knockdown (KD) female was born that subsequently produced 80 piglets from 6 litters with 46 hemizygous progeny (57% transgenic). Hemizygous GNRHR2 KD (n = 10) and littermate control (n = 7) males were monitored at 40, 100, 150, 190, 225 and 300 days of age; body weight and testis size were measured and serum was isolated and assayed for testosterone and luteinizing hormone (LH) concentrations. Body weight of GNRHR2 KD boars was not different from littermate controls (P = 0.14), but testes were smaller (P < 0.05; 331.8 vs. 374.8 cm3, respectively). Testosterone concentrations tended (P = 0.06) to be reduced in GNRHR2 KD (1.6 ng/ml) compared to littermate control (4.2 ng/ml) males, but LH levels were similar (P = 0.47). The abundance of GNRHR2 mRNA was reduced (P < 0.001) by 69% in testicular tissue from mature GNRHR2 KD (n = 5) versus littermate control (n = 4) animals. These swine represent the first genetically-engineered model to elucidate the function of GNRH2 and its receptor in mammals.
Subject(s)
Animals, Genetically Modified/genetics , Gonadotropin-Releasing Hormone/genetics , Receptors, LHRH/genetics , Animals , Animals, Genetically Modified/growth & development , Female , Gene Expression Regulation , Gene Knockdown Techniques , Gonadotropin-Releasing Hormone/biosynthesis , Hemizygote , Luteinizing Hormone/blood , Male , RNA, Small Interfering/genetics , Receptors, LHRH/biosynthesis , Swine/genetics , Swine/growth & development , Testis/growth & development , Testis/metabolism , Testosterone/bloodABSTRACT
This study was performed to explain how the molecular processes governing the biosynthesis of gonadotropin-releasing hormone (GnRH) and GnRH receptor (GnRHR) in the hypothalamic-pituitary unit are reflected by luteinizing hormone (LH) secretion in sheep during anoestrous period and during luteal and follicular phases of the oestrous cycle. Using an enzyme-linked immunosorbent assay (ELISA), we analyzed the levels of GnRH and GnRHR in preoptic area (POA), anterior (AH) and ventromedial hypothalamus (VM), stalk-median eminence (SME), and GnRHR in the anterior pituitary gland (AP). Radioimmunoassay has also been used to define changes in plasma LH concentrations. The study provides evidence that the levels of GnRH in the whole hypothalamus of anoestrous ewes were lower than that in sheep during the follicular phase of the oestrous cycle (POA: p < 0.001, AH: p < 0.001, VM: p < 0.01, SME: p < 0.001) and not always than in luteal phase animals (POA: p < 0.05, SME: p < 0.05). It has also been demonstrated that the GnRHR amount in the hypothalamus-anterior pituitary unit, as well as LH level, in the blood in anoestrous ewes were significantly lower than those detected in animals of both cyclic groups. Our data suggest that decrease in LH secretion during the long photoperiod in sheep may be due to low translational activity of genes encoding both GnRH and GnRHR.
Subject(s)
Anestrus/metabolism , Estrous Cycle/metabolism , Gonadotropin-Releasing Hormone/biosynthesis , Hypothalamo-Hypophyseal System/metabolism , Receptors, LHRH/biosynthesis , Anestrus/blood , Animals , Anterior Hypothalamic Nucleus/metabolism , Estrous Cycle/blood , Female , Luteinizing Hormone/blood , Median Eminence/metabolism , Pituitary Gland/metabolism , Preoptic Area/metabolism , Sheep , Ventromedial Hypothalamic Nucleus/metabolismABSTRACT
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 animal's 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.
Subject(s)
Gonadotropin-Releasing Hormone/biosynthesis , Hypothalamus/physiology , Pituitary Gland/physiology , Receptors, LHRH/biosynthesis , Sheep/physiology , Stress, Physiological/physiology , Animals , Estrous Cycle/physiology , Female , Gene Expression Regulation/physiology , Luteinizing Hormone , Pregnancy , Time FactorsABSTRACT
No disponible
Subject(s)
Humans , Female , Neurotransmitter Agents/analysis , Neurotransmitter Agents/history , Gonadotropin-Releasing Hormone/analysis , Gonadotropin-Releasing Hormone/history , Receptors, LHRH/administration & dosage , Receptors, LHRH/antagonists & inhibitors , Receptors, LHRH/biosynthesis , Receptors, Neuropeptide/administration & dosage , Receptors, Neuropeptide/therapeutic useABSTRACT
OBJECTIVE: To investigate whether gonadotropin-releasing hormone (GnRH) and GnRH receptor (GnRHR) are expressed at tubal ectopic pregnancy sites, and to study the potential role of GnRH signaling in regulating immortalized human trophoblast cell viability. DESIGN: Immunohistochemical and experimental studies. SETTING: Academic research laboratory. PATIENT(S): Fallopian tube implantation sites (n = 25) were collected from women with ectopic pregnancy. First-trimester human placenta biopsies (n = 5) were obtained from elective terminations of pregnancy. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): GnRH and GnRHR expression was examined by means of immunohistochemistry and histoscoring. Trophoblastic BeWo choriocarcinoma and immortalized extravillous trophoblast (HTR-8/SVneo) cell viability was examined by means of cell counting after incubation with GnRH and/or GnRH antagonist (Antide). RESULT(S): GnRH and GnRHR immunoreactivity was detected in cytotrophoblast, syncytiotrophoblast, and extravillous trophoblast in all women with tubal pregnancy. GnRH immunoreactivity was higher and GnRHR immunoreactivity lower in syncytiotrophoblast compared with cytotrophoblast. GnRH and GnRHR immunoreactivity was detected in adjacent fallopian tube epithelium. Whereas neither GnRH nor Antide altered HTR-8/SVneo cell viability, treatment with GnRH significantly increased the overall cell viability of BeWo cells at 48 and 72 hours, and these effects were abolished by pretreatment with Antide. CONCLUSION(S): GnRH and GnRHR are expressed in trophoblast cell populations and fallopian tube epithelium at tubal ectopic pregnancy sites. GnRH increases BeWo cell viability, an effect mediated by the GnRHR. Further work is required to investigate the potential role of GnRH signaling in ectopic pregnancy.
Subject(s)
Embryo Implantation/physiology , Gene Expression Regulation, Developmental , Gonadotropin-Releasing Hormone/biosynthesis , Pregnancy, Tubal/metabolism , Receptors, LHRH/biosynthesis , Cell Line, Transformed , Cell Line, Tumor , Cell Survival/physiology , Fallopian Tubes/metabolism , Fallopian Tubes/pathology , Female , Humans , Pregnancy , Pregnancy, Tubal/pathology , Trophoblasts/metabolism , Trophoblasts/pathologyABSTRACT
In vertebrates, gonadotropin-releasing hormone (GnRH) and gonadotropin-inhibitory hormone (GnIH), respectively, regulate reproduction in positive and negative manners. GnIH belongs to the LPXRFa family of peptides previously identified in mammalian and nonmammalian vertebrates. Studying the detailed distribution of LPXRFa as well as its receptor (LPXRFa-R) in the brain and pituitary is important for understanding their multiple action sites and potential functions. However, the distribution of LPXRFa and LPXRFa-R has not been studied in teleost species, partially because of the lack of fish-specific antibodies. Therefore, in the present study, we generated specific antibodies against LPXRFa and its receptor from Nile tilapia (Oreochromis niloticus), and examined their distributions in the brain and pituitary by immunohistochemistry. Tilapia LPXRFa-immunoreactive neurons lie in the posterior ventricular nucleus of the caudal preoptic area, whereas LPXRFa-R-immunoreactive cells are distributed widely. Double immunofluorescence showed that neither LPXRFa-immunoreactive fibers nor LPXRFa-R is closely associated or coexpressed with GnRH1, GnRH3, or kisspeptin (Kiss2) neurons. In the pituitary, LPXRFa fibers are closely associated with gonadotropic endocrine cells [expressing luteinizing hormone (LH) and follicle-stimulating hormone (FSH)], with adrenocorticomelanotropic cells [corticotropin (ACTH) and α-melanotropin (α-MSH)], and with somatolactin endocrine cells. In contrast, LPXRFa-R are expressed only in LH, ACTH, and α-MSH cells. These results suggest that LPXRFa and LPXRFa-R signaling acts directly on the pituitary cells independent from GnRH or kisspeptin and could play multiple roles in reproductive and nonreproductive functions in teleosts. J. Comp. Neurol. 524:2753-2775, 2016. © 2016 Wiley Periodicals, Inc.
Subject(s)
Brain Chemistry , Gonadotropin-Releasing Hormone/analysis , Hypothalamic Hormones/analysis , Pituitary Gland/chemistry , Receptors, Gonadotropin/analysis , Receptors, LHRH/analysis , Animals , Brain/metabolism , Brain Chemistry/physiology , Gonadotropin-Releasing Hormone/biosynthesis , Hypothalamic Hormones/biosynthesis , Male , Pituitary Gland/metabolism , Receptors, Gonadotropin/biosynthesis , Receptors, LHRH/biosynthesis , TilapiaABSTRACT
Growing evidence has revealed the deleterious influence of environmental and food contaminants on puberty onset and development in both animals and children, provoking an increasing health concern. T-2 toxin, a naturally-produced Type A trichothecene mycotoxin which is frequently found in cereal grains and products intended for human and animal consumption, has been shown to impair the reproduction and development in animals. Nevertheless, whether this trichothecene mycotoxin can disturb the onset of puberty in females remains unclear. To clarify this point, infantile female rats were given a daily intragastric administration of vehicle or 187.5 µg/kg body weight of T-2 toxin for five consecutive days from postnatal day 15 to 19, and the effects on puberty onset were evaluated in the present study. The results revealed that the days of vaginal opening, first dioestrus, and first estrus in regular estrous cycle were delayed following prepubertal exposure to T-2 toxin. The relative weights of reproductive organs uterus, ovaries, and vagina, and the incidence of corpora lutea were all diminished in T-2 toxin-treated rats. Serum levels of gonadotropins luteinizing hormone, follicle-stimulating hormone, and estradiol were also reduced by T-2 toxin treatment. The mRNA expressions of hypothalamic gonadotropin-releasing hormone (GnRH) and pituitary GnRH receptor displayed significant reductions following exposure to T-2 toxin, which were consistent with the changes of serum gonadotropins, delayed reproductive organ development, and delayed vaginal opening. In conclusion, the present study reveals that prepubertal exposure to T-2 toxin delays the onset of puberty in immature female rats, probably by the mechanism of disturbance of hypothalamic-pituitary-gonadal (HPG) axis function. Considering the vulnerability of developmental children to food contaminants and the relative high level of dietary intake of T-2 toxin in children, we think the findings of the present study provide valuable information for the health risk assessment in children.
Subject(s)
Sexual Maturation/drug effects , T-2 Toxin/toxicity , Animals , Body Weight/drug effects , Corpus Luteum/drug effects , Diestrus/drug effects , Estrous Cycle/drug effects , Estrus/drug effects , Female , Gonadotropin-Releasing Hormone/blood , Intubation, Gastrointestinal , Organ Size , Ovary/drug effects , Ovary/growth & development , Rats , Rats, Sprague-Dawley , Receptors, LHRH/biosynthesis , Receptors, LHRH/drug effects , T-2 Toxin/administration & dosage , Uterus/drug effects , Uterus/growth & development , Vagina/drug effects , Vagina/growth & developmentABSTRACT
DNA cis-acting elements involved in gene regulation may actively contribute to adaptation processes because they are submitted to lower evolutionary constraints than coding DNA. In this regard, comparisons of the mechanisms underlying basal and regulated Gnrhr expression have revealed some features that promote stable and consistent Gnrhr expression in pituitary gonadotroph cells in different species. The presence of two divergent SF1 (NR5A1) response elements in all analysed mammalian Gnrhr promoters probably comprises one of the features that ensures reliable expression in the pituitary. By contrast, in other tissues, such as the hippocampus and testis, our analyses revealed dissimilar levels of Gnrhr expression among species. Indeed, Gnrhr was consistently expressed after birth in the rat but not the mouse hippocampus. Similar discrepancies were observed in foetal and adult testes. The ability of the rat promoter to drive reporter gene expression in the hippocampus and testis of transgenic mice just as it naturally directs the expression of the endogenous Gnrhr in rats strongly suggests that regulatory DNA sequences contained species-specific instructions prevailing over other controls. The major conclusion emerging from these studies is that Gnrhr promoter sequences are mainly responsible for directing transcriptional programmes and play a predominant role over the species-specific cell environment.
Subject(s)
Biological Evolution , Gene Expression Regulation/genetics , Pituitary Gland/metabolism , Promoter Regions, Genetic/genetics , Receptors, LHRH/biosynthesis , Animals , Hippocampus/metabolism , Male , Mice , Rats , Species Specificity , Testis/metabolismABSTRACT
Testis growth during early life is important for future male fertility and shows acceleration during the first months of life in humans. This acceleration coincides with the peak in gonadotropic hormones in the blood, while the role of hypothalamic factors remains vague. Using neonatal rats to assess this issue, we found that day 9 of life is likely critical for testis development in rats. Before this day, testicular growth was proportional to body weight gain, but after that the testes showed accelerated growth. Hypothalamic kisspeptin and its receptor mRNA levels begin to elevate 2 days later, at day 11. A significant increase in the mRNA levels for gonadotropin-releasing hormone (GnRH) receptors in the hypothalamus between days 5 and 7 was followed by a 3-fold decrease in GnRH mRNA levels in this brain region during the next 2 days. Starting from day 9, hypothalamic GnRH mRNA levels increased significantly and positively correlated with accelerated testicular growth. Triptorelin, an agonist of GnRH, at a dose that had no effect on testicular growth during "proportional" period, increased testis weights during the period of accelerated growth. The insensitivity of testicular growth to GnRH during "proportional" period was supported by inability of a 2.5-fold siRNA knockdown of GnRH expression in the hypothalamus of the 7-day-old animals to produce any effect on their testis weights. GnRH receptor blockade with cetrorelix was also without effect on testis weights during "proportional" period but the same doses of this GnRH antagonist significantly inhibited "accelerated" testicular growth. GnRH receptor mRNA levels in the pituitary as well as plasma LH concentrations were higher during "accelerated" period of testicular growth than during "proportional" period. In general, our data defined two distinct periods in rat testicular development that are primarily characterized by different responses to GnRH signaling.
Subject(s)
Aging/physiology , Gonadal Hormones/metabolism , Hypothalamus/metabolism , Neurosecretory Systems/growth & development , Testis/growth & development , Aging/drug effects , Animals , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Kisspeptins/metabolism , Luteolytic Agents/pharmacology , Male , RNA, Messenger/biosynthesis , Rats , Rats, Wistar , Receptors, LHRH/biosynthesis , Signal Transduction , Triptorelin Pamoate/pharmacologyABSTRACT
Two novel small molecule gonadotropin-releasing hormone (GnRH) receptor antagonists (12 and 13) of the furamide-class were synthesized and evaluated in vitro for their receptor binding affinities for the rat GnRH receptor. Radiolabeling with no carrier added fluorine-18 of the appropriate precursors was investigated in a one-step reaction. LogP (Octanol/PBS pH 7.4) and serum stability of the compounds were investigated. The antagonists showed low nM affinity for the rat GnRH receptor. (18)F-radiolabled compounds were obtained in high radiochemical purity (>95%) and specific activity (>75 GBq/µmol). These findings suggest this class of compounds holds promise as potential probes for PET targeting of GnRH-receptor expression.
Subject(s)
Positron-Emission Tomography , Radiopharmaceuticals/pharmacology , Receptors, LHRH/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Animals , Dose-Response Relationship, Drug , Fluorine Radioisotopes/chemistry , HEK293 Cells , Humans , Molecular Structure , Radiopharmaceuticals/chemical synthesis , Radiopharmaceuticals/chemistry , Rats , Receptors, LHRH/biosynthesis , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/chemistry , Structure-Activity RelationshipABSTRACT
OBJECTIVE: Advanced or recurrent endometrial cancer (EC) no longer amenable to surgery or radiotherapy is a life-threatening disease with limited therapeutic options left. Eighty percent of ECs express receptors for luteinizing hormone-releasing hormone (LHRH), which can be targeted by AEZS-108 (zoptarelin doxorubicin acetate). This phase 2 trial was performed to assess the efficacy and safety of AEZS-108 in this group of patients. METHODS: Patients had FIGO (Fédération Internationale de Gynécologie et d'Obstétrique) III or IV or recurrent EC, LHRH receptor-positive tumor status, and at least had 1 measurable lesion (Response Evaluation Criteria in Solid Tumors). Prior anthracycline therapy was not allowed. Patients received AEZS-108 as a 2-hour infusion on day 1 of a 21-day cycle. The treatment was continued for a maximum of 6 to 8 cycles. The primary end point was the response rate determined by the Response Evaluation Criteria in Solid Tumors. RESULTS: From April 2008 to November 2009, 44 patients were included in the study at 8 centers in Germany (AGO) and 3 centers in Bulgaria. Forty-three of these patients were eligible. Two (5%) patients had a complete remission, and 8 (18%) achieved a partial remission. Stable disease for at least 6 weeks was observed in 44%. The median time to progression was 7 months, and the median overall survival was 15 months. The most frequently reported grade 3 or 4 adverse effects were neutropenia (12%) and leucopenia (9%). CONCLUSIONS: AEZS-108, an LHRH-agonist coupled to doxorubicin, has significant activity and low toxicity in women with advanced or recurrent LHRH receptor-positive EC, supporting the principle of receptor-mediated targeted chemotherapy.
Subject(s)
Doxorubicin/analogs & derivatives , Endometrial Neoplasms/drug therapy , Gonadotropin-Releasing Hormone/analogs & derivatives , Neoplasm Recurrence, Local/drug therapy , Receptors, LHRH/antagonists & inhibitors , Adult , Aged , Aged, 80 and over , Doxorubicin/pharmacology , Doxorubicin/therapeutic use , Endometrial Neoplasms/metabolism , Female , Gonadotropin-Releasing Hormone/pharmacology , Gonadotropin-Releasing Hormone/therapeutic use , Humans , Middle Aged , Neoplasm Recurrence, Local/metabolism , Receptors, LHRH/biosynthesisABSTRACT
Uveal melanoma is the most common primary intraocular malignancy in adults, with a very high mortality rate due to frequent liver metastases. Consequently, the therapy of uveal melanoma remains a major clinical challenge and new treatment approaches are needed. For improving diagnosis and designing a rational and effective therapy, it is essential to elucidate molecular characteristics of this malignancy. The aim of this study therefore was to evaluate as a potential therapeutic target the expression of luteinizing hormone-releasing hormone (LHRH) receptor in human uveal melanoma. The expression of LHRH ligand and LHRH receptor transcript forms was studied in 39 human uveal melanoma specimens by RT-PCR using gene specific primers. The binding charachteristics of receptors for LHRH on 10 samples were determined by ligand competition assays. The presence of LHRH receptor protein was further evaluated by immunohistochemistry. The expression of mRNA for type I LHRH receptor was detected in 18 of 39 (46%) of tissue specimens. mRNA for LHRH-I ligand could be detected in 27 of 39 (69%) of the samples. Seven of 10 samples investigated showed high affinity LHRH-I receptors. The specific presence of full length LHRH receptor protein was further confirmed by immunohistochemistry. A high percentage of uveal melanomas express mRNA and protein for type-I LHRH receptors. Our results support the merit of further investigation of LHRH receptors in human ophthalmological tumors. Since diverse analogs of LHRH are in clinical trials or are already used for the treatment of various cancers, theseanalogs could be considered for the LHRH receptor-based treatment of uveal melanoma.
Subject(s)
Melanoma/metabolism , Receptors, LHRH/biosynthesis , Uveal Neoplasms/metabolism , Adult , Aged , Aged, 80 and over , Female , Humans , Immunohistochemistry , Male , Melanoma/genetics , Middle Aged , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Receptors, LHRH/genetics , Uveal Neoplasms/geneticsABSTRACT
PURPOSE: The proposed project is aimed at enhancing the efficiency of epithelial ovarian cancer treatment and reducing adverse side effects of chemotherapy using nanotechnology. Overexpression of the CD44 membrane receptor results in tumor initiation, growth, cancer stem cells' specific behavior, development of drug resistance, and metastases. We hypothesize that a developed cancer-targeted delivery system that combines CD44 siRNA with paclitaxel would successfully deliver its payload inside cancer cells, effectively induce cell death, and prevent metastases. EXPERIMENTAL DESIGN: We synthesized, characterized, and tested a nanoscale-based drug delivery system (DDS) containing a modified polypropylenimine (PPI) dendrimer as a carrier; anticancer drug paclitaxel as a cell death inducer; a synthetic analog of luteinizing hormone-releasing hormone (LHRH) peptide as a tumor-targeting moiety; and siRNA targeted to CD44 mRNA. The proposed DDS was tested in vitro and in vivo using metastatic ovarian cancer cells isolated from patients with malignant ascites. RESULTS: We found that in contrast with cells isolated from primary tumors, CD44 was highly overexpressed in metastatic cancer cells. Treatment with the proposed tumor-targeted nanoscale-based nucleic acid and DDS led to the suppression of CD44 mRNA and protein, efficient induction of cell death, effective tumor shrinkage, and prevention of adverse side effects on healthy organs. CONCLUSION: We show a high therapeutic potential for combinatorial treatment of ovarian carcinoma with a novel DDS that effectively transports siRNA targeting to CD44 mRNA simultaneously with cytotoxic agents. Clin Cancer Res; 19(22); 6193-204. ©2013 AACR.
Subject(s)
Ascitic Fluid/cytology , Dendrimers/pharmacology , Ovarian Neoplasms/drug therapy , Paclitaxel/pharmacology , RNA, Small Interfering/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Biological Transport , Female , Gene Expression , Gonadotropin-Releasing Hormone/analogs & derivatives , Gonadotropin-Releasing Hormone/biosynthesis , Humans , Hyaluronan Receptors/biosynthesis , Hyaluronan Receptors/genetics , Mice , Mice, Nude , Nanomedicine , Nanoparticles/chemistry , Neoplasm Invasiveness , Neoplasm Transplantation , Paclitaxel/administration & dosage , Pilot Projects , Polypropylenes , RNA Interference , RNA, Small Interfering/administration & dosage , Receptors, LHRH/biosynthesis , Transplantation, Heterologous , Tumor Cells, CulturedABSTRACT
Several evidences supported the existence of melatonin effects on reproductive system in fish. In order to investigate whether melatonin is involved in the modulation of GnRH systems in the European sea bass, we have injected melatonin (0.5 µg/g body mass) in male specimens. The brain mRNA transcript levels of the three GnRH forms and the five GnRH receptors present in this species were determined by real time quantitative PCR. Our findings revealed day-night variations in the brain expression of GnRH-1, GnRH-3 and several GnRH receptors (dlGnRHR-II-1c, -2a), which exhibited higher transcript levels at mid-light compared to mid-dark phase of the photocycle. Moreover, an inhibitory effect of melatonin on the nocturnal expression of GnRH-1, GnRH-3, and GnRH receptors subtypes 1c, 2a and 2b was also demonstrated. Interestingly, the inhibitory effect of melatonin affected the expression of hypophysiotrophic GnRH forms and GnRH receptors that exhibit day-night fluctuations, suggesting that exogenous melatonin reinforce physiological mechanisms already established. These interactions between melatoninergic and GnRH systems could be mediating photoperiod effects on reproductive and other rhythmic physiological events in the European sea bass.