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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., (© 2022. The Author(s).)

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., (Copyright © 2019 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.)

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 2038 bp in length, including an open reading frame (ORF) of 1566 bp, a 57 bp 5'-untranslated region (5'-UTR) and a 415 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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (© 2018 Wiley Periodicals, Inc.)

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., (Copyright © 2018 IMSS. Published by Elsevier Inc. All rights reserved.)

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.

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 cm 3 , 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.

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.

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (© 2016 Wiley Periodicals, Inc.)

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., (Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

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.

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., (© 2015 British Society for Neuroendocrinology.)

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.

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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.

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.

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.

Pheochromocytoma is a rare but potentially lethal chromaffin cell tumor with currently no effective treatment. Peptide hormone receptors are frequently overexpressed on endocrine tumor cells and can be specifically targeted by various anti-tumor peptide analogs. The present study carried out on mouse pheochromocytoma cells (MPCs) and a more aggressive mouse tumor tissue-derived (MTT) cell line revealed that these cells are characterized by pronounced expression of the somatostatin receptor 2 (sst2), growth hormone-releasing hormone (GHRH) receptor and the luteinizing hormone-releasing hormone (LHRH) receptor. We further demonstrated significant anti-tumor effects mediated by cytotoxic somatostatin analogs, AN-162 and AN-238, by LHRH antagonist, Cetrorelix, by the cytotoxic LHRH analog, AN-152, and by recently developed GHRH antagonist, MIA-602, on MPC and for AN-152 and MIA-602 on MTT cells. Studies of novel anti-tumor compounds on these mouse cell lines serve as an important basis for mouse models of metastatic pheochromocytoma, which we are currently establishing., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

To determine whether hormone-receptor signaling pathways in the thymus are altered by active immunization against gonadotrophin-releasing hormone I (GnRH), 3-week-old Sprague-Dawley male rats received GnRH-tandem-OVA peptides (200 μg/ml), and the effects were compared to a control group. Serum testosterone, LH and FSH concentrations were markedly reduced, with severe testicular atrophy, compared to controls, demonstrating effective blockade of the pituitary-gonadal axis. The reduction in LH and FSH concentrations in the thymus of immunized animals was lower than that observed in the serum, where a significant difference (P<0.001) in concentration was observed between both groups. Concentrations of GnRH were increased in the thymus of immunized rats. In thymic tissue, GnRHR, FSHR and LHR demonstrated stronger immunostaining, and AR weaker staining, in the immunized group compared to controls. Reproductive hormone receptor mRNA expression was consistent with protein variations in the immunized thymus. Compared to controls, GnRHR gene levels were significantly increased (P<0.05), however, AR mRNA expression were greatly decreased with immune week-age (P<0.05). Both FSHR and LHR mRNA expression levels were significantly higher in the treated group than in controls in the first three samples (P<0.05). When GnRHR was blocked by an antagonist in thymocytes, all reproductive hormone receptor gene expressions were significantly increased (P<0.001). In summary, these findings suggest that active immunization against GnRH can up-regulate GnRH receptor and gonadotropin receptor signaling, by stimulating thymic autocrine and paracrine function, whereas the androgen receptor is down-regulated due to a lack of testosterone secretion in the thymus., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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.

Gonadotropin-releasing hormone (GnRH) agonists are widely used for the treatment of advanced prostate cancer (PCa). Agonists activate the GnRH receptor (GnRH-R), triggering apoptosis in PCa cells. In gonadotropes, the amount of GnRH-R in the plasma membrane is regulated by protein folding and endoplasmic reticulum retention, mechanisms that can be overcome by the pharmacoperone IN3. Our aim was to describe the intracellular distribution of GnRH-R in PCa cells and its relation to response to GnRH analog treatments. The expressions of GnRH-R in PCa biopsies were evaluated by immunohistochemistry and the intracellular distribution was determined by immunofluorescence in primary cell cultures from human PCa samples. Cultured cells were pretreated with IN3 and then with leuprolide. Cell survival was evaluated by 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) thiazolyl blue formazan and cell cycle and apoptosis by flow cytometry. We observed that the expression of GnRH-R decreased according to malignant progression. Most GnRH-R are located inside the cell, colocalizing with endoplasmic reticulum markers. The treatment with IN3 decreased cellular GnRH-R retention, increasing plasma membrane expression in approximately 60%. Pretreatment with IN3 decreased PCa cell survival compared with leuprolide-alone treatment, primarily because of an increase in apoptosis. We conclude that the response of PCa cells to leuprolide is related to the amount of GnRH-R in the plasma membrane. Therefore, pretreatment evaluation of the amount of these receptors may be a predictor of the outcome of leuprolide treatment in PCa patients. Assessment of systemic IN3 effect would be necessary to determine its utility as an adjuvant treatment in hormone-resistant tumors.

Gonadotropin-releasing hormone (either GnRH1 or GnRH2) exerts a local activity in vertebrate testis, including human testis. Relationships between endocannabinoid (eCB) and GnRH systems in gonads have never been elucidated in any species so far. To reveal a cross-talk between eCBs and GnRH at testicular level, we characterized the expression of GnRH (GnRH1 and GnRH2) as well as GnRH receptor (GnRH-R1, -R2, and -R3) mRNA in the testis of the anuran amphibian Rana esculenta during the annual sexual cycle; furthermore, the corresponding transcripts were localized inside the testis by in situ hybridization. The possible endogenous production of the eCB, anandamide (AEA), was investigated in testis by analyzing the expression of its biosynthetic enzyme, Nape-pld. Incubations of testis pieces with AEA were carried out in the postreproductive period (June) and in February, when a new spermatogenetic wave takes place. In June, AEA treatment significantly decreased GnRH1 and GnRH-R2 mRNA, stimulated the transcription of GnRH2 and GnRH-R1, and did not affect GnRH-R3 expression. In February, AEA treatment upregulated GnRH2 and GnRH-R3 mRNA, downregulated GnRH-R2, and did not affect GnRH1 and GnRH-R1 expression. These effects were mediated by type 1 cannabinoid receptor (CB1) since they were fully counteracted by SR141716A (Rimonabant), a selective CB1 antagonist. In conclusion, eCB system modulates GnRH activity in frog testis during the annual sexual cycle in a stage-dependent fashion.

Feeding behavior and reproduction are coordinately regulated by the brain via neurotransmitters, circulating hormones, and neuropeptides. Reduced feeding allows animals to engage in other behaviors important for fitness, including mating and parental care. Some fishes cease feeding for weeks at a time in order to provide care to their young by brooding them inside the male or female parent's mouth. Maternal mouthbrooding is known to impact circulating hormones and subsequent reproductive cycles, but neither the full effects of food deprivation nor the neural mechanisms are known. Here we ask what effects mouthbrooding has on several physiological processes including gonad and body mass, brain neuropeptide and receptor gene expression, and circulating steroid hormones in a mouthbrooding cichlid species, Astatotilapia burtoni. We ask whether any observed changes can be explained by food deprivation, and show that during mouthbrooding, ovary size and circulating levels of androgens and estrogens match those seen during food deprivation. Levels of gonadotropin-releasing hormone 1 (GnRH1) mRNA in the brain were low in food-deprived females compared to controls and in mouthbrooding females compared to gravid females. Levels of mRNA encoding two peptides involved in regulating feeding, hypocretin and cholecystokinin, were increased in the brains of food-deprived females. Brain mRNA levels of two receptors, GnRH receptor 2 and NPY receptor Y8c, were elevated in mouthbrooding females compared to the fed condition, but NPY receptor Y8b mRNA was differently regulated by mouthbrooding. These results suggest that many, but not all, of the characteristic physiological changes that occur during mouthbrooding are consequences of food deprivation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

This study is to investigate the anti-tumor effect in vitro of methotrexate modified by LH-RH peptide (LH-RH-MTX). LH-RH receptors highly expressing MCF-7 human breast carcinoma cell line and lowly expressing K562 human erythroleukemia cell line were served as the tested cells. The cell proliferation inhibition rates of LH-RH-MTX were detected by MTT colorimetric assay. The effects of LH-RH-MTX on the cell cycle and apoptosis rates were detected by flow cytometry. The inhibition rate of LH-RH-MTX on MCF-7 cells was much higher than that on K562 cells, and the inhibition rate of LH-RH-MTX on MCF-7 cells was much higher than that of free MTX at the same concentration. The inhibition rate of LH-RH-MTX on rat bone marrow mononuclear cells was less than that of free MTX. The number of MCF-7 cells in S phase increased after administration of LH-RH-MTX. The apoptosis rate of LH-RH-MTX group significantly increased compared with that of the control group and MTX group. The relative expression of LHRHR mRNA of LH-RH-MTX group markedly decreased compared with that of the control group and MTX group. LH-RH-MTX is realizable to reduce drug side effects, increase the therapeutic index and achieve tumor-targeted therapy.

Estrogens have profound effects on the serum and anterior pituitary (AP) insulin-like growth factor (IGF) system in pigs. In this study we determined whether administration of the phytoestrogen genistein increased serum and AP concentrations of IGF-I and relative amounts of serum and AP insulin-like growth factor-binding protein (IGFBP). Twenty barrows of similar age (190 d) and weight (110 kg) were stratified by litter into one of four treatments: controls (C), estradiol (E), 200 mg genistein (G200), and 400 mg genistein (G400). Estradiol-treated pigs were injected daily with 2 mg of estradiol-17β intramuscularly (i.m.), whereas the G200 and G400 groups were injected daily with either 200 or 400 mg of genistein i.m., respectively, beginning on d 0 and continuing through d 15. Blood was collected on d 0, 3, 6, 9, and 13. Blood and AP were collected at slaughter on d 16. Serum and AP concentrations of IGF-I and luteinizing hormone (LH) were determined by radioimmunoassay. Relative amounts of serum IGFBP were determined by Western ligand blot analysis. Relative expression of AP IGF-I, IGF-I receptor (IGF-IR), gonadotropin-releasing hormone receptor, and LHβ subunit was determined by real-time reverse transcriptase polymerase chain reaction. Anterior pituitary concentrations of IGF-I were greater (P > 0.05) in E and G400 pigs compared with controls, whereas AP concentrations of LH were greater (P < 0.05) in G400 pigs compared with C and G200 pigs. Relative expression of LHβ was greater in G200 pigs compared with C pigs but did not differ from that in G400 pigs. Relative expression of AP IGF-IR was greater (P < 0.05) in E pigs compared with all other treatments; however, relative expression of AP IGF-IR was greater (P < 0.05) in both G200 and G400 pigs vs C pigs. No differences were detected (P > 0.05) in serum concentrations of IGF-I or relative amounts of serum and AP IGFBP among treatments. These data provide evidence that genistein is capable of modulating components of the AP IGF system that could affect the synthesis and release of LH., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) is present in high concentrations within the hypothalamus, suggesting that it may be a hypophysiotropic factor, whereas pituitary expression suggests a paracrine function. PACAP stimulates gonadotropin secretion and enhances GnRH responsiveness. PACAP increases gonadotropin α-subunit (αGSU), lengthens LHβ, but reduces FSHβ mRNA levels in adult pituitary cell cultures in part by increasing follistatin. PACAP stimulates LH secretion in rats; however, acceptance of PACAP as a regulator of reproduction has been limited by a paucity of in vivo studies. We created a transgenic mouse model of pituitary PACAP overexpression using the αGSU subunit promoter. Real-time PCR was used to evaluate PACAP, follistatin, GnRH receptor, and the gonadotropin subunit mRNA in male transgenic and wild-type mice of various ages. Transgenic mice had greater than 1000-fold higher levels of pituitary PACAP mRNA; and immunocytochemistry, Western blot, and ELISA analyses confirmed high peptide levels. FSH, LH, and testosterone levels were significantly suppressed, and the timing of puberty was substantially delayed in PACAP transgenic mice in which gonadotropin subunit and GnRH receptor mRNA levels were reduced and pituitary follistatin expression was increased. Microarray analyses revealed 1229 of 45102 probes were significantly (P < 0.01) different in pituitaries from PACAP transgenic mice, of which 83 genes were at least 2-fold different. Genes involved in small molecule biochemistry, cancer, and reproductive system diseases were the top associated networks. The GnRH signaling pathway was the top canonical pathway affected by pituitary PACAP excess. These experiments provide the first evidence that PACAP affects gonadotropin expression and sexual maturation in vivo.

In the course of evolution, social behavior has been a strikingly potent selective force in shaping brains to control action. Physiological, cellular, and molecular processes reflect this evolutionary force, particularly in the regulation of reproductive behavior and its neural circuitry. Typically, experimental analysis is directed at how the brain controls behavior, but the brain is also changed by behavior over evolution, during development, and through its ongoing function. Understanding how the brain is influenced by behavior offers unusual experimental challenges. General principles governing the social regulation of the brain are most evident in the control of reproductive behavior. This is most likely because reproduction is arguably the most important event in an animal's life and has been a powerful and essential selective force over evolution. Here I describe the mechanisms through which behavior changes the brain in the service of reproduction using a teleost fish model system.

Background and Aims: Human metastatic prostate cancer cell growth can be inhibited by GnRH analogs but effects on virus-immortalized prostate cells have not been investigated., Methods: Virus-immortalized prostate cells were stably transfected with rat GnRH receptor cDNA and levels of GnRH binding were correlated with GnRH effects on signaling, cell cycle, growth, exosome production, and apoptosis., Results: High levels of cell surface GnRH receptor occurred in transfected papillomavirus-immortalized WPE-1-NB26 epithelial cells but not in non-tumourigenic RWPE-1, myoepithelial WPMY-1 cells, or SV40-immortalized PNT1A. Endogenous cell surface GnRH receptor was undetectable in non-transfected cells or cancer cell lines LNCaP, PC3, and DU145. GnRH receptor levels correlated with induction of inositol phosphates, elevation of intracellular Ca(2+) , cytoskeletal actin reorganization, modulation of ERK activation and cell growth-inhibition with GnRH agonists. Hoechst 33342 DNA staining-cell sorting indicated accumulation of cells in G2 following agonist treatment. Release of exosomes from transfected WPE-1-NB26 was unaffected by agonists, unlike induction observed in HEK293([SCL60]) cells. Increased PARP cleavage and apoptotic body production were undetectable during growth-inhibition in WPE-1-NB26 cells, contrasting with HEK293([SCL60]) . EGF receptor activation inhibited GnRH-induced ERK activation in WPE-1-NB26 but growth-inhibition was not rescued by EGF or PKC inhibitor Ro320432. Growth of cells expressing low levels of GnRH receptor was not affected by agonists., Conclusions: Engineered high-level GnRH receptor activation inhibits growth of a subset of papillomavirus-immortalized prostate cells. Elucidating mechanisms leading to clone-specific differences in cell surface GnRH receptor levels is a valuable next step in developing strategies to exploit prostate cell anti-proliferation using GnRH agonists., (Copyright © 2010 Wiley-Liss, Inc.)

Background: The majority of men will develop symptoms of benign prostatic hyperplasia (BPH) after 70 years of age. Various studies indicate that antagonists of LHRH, such as cetrorelix, exert direct inhibitory effects on BPH mediated by specific LHRH receptors. Our aim was to investigate the mRNA for LHRH and LHRH receptors and the expression of LHRH receptors in specimens of human BPH., Methods: The expression of mRNA for LHRH (n=35) and LHRH receptors (n=55) was investigated by RT-PCR in surgical specimens of BPH, using specific primers. The characteristics of binding sites for LHRH on 20 samples were determined by ligand competition assays. The LHRH receptor expression was also examined in 64 BPH specimens by immunohistochemistry., Results: PCR products for LHRH were found in 18 of 35 (51%) BPH tissues and mRNA for LHRH receptors was detected in 39 of 55 (71%) BPH specimens. Eighteen of 20 (90%) samples showed a single class of high affinity binding sites for [D-Trp(6) ]LHRH with a mean K(d) of 4.04 nM and a mean B(max) of 527.6 fmol/mg membrane protein. LHRH antagonist cetrorelix showed high affinity binding to LHRH receptors in BPH. Positive immunohistochemical reaction for LHRH receptors was present in 42 of 64 (67%) BPH specimens., Conclusion: A high incidence of LHRH receptors in BPH supports the use of LHRH antagonists such as cetrorelix, for treatment of patients with lower urinary tract symptoms from BPH., (Copyright © 2010 Wiley-Liss, Inc.)

Neuromodulators including gonadotropin-releasing hormone (GnRH) and sex steroids help integrate an animal's internal physiological state with incoming external cues, and can have profound effects on the processing of behaviorally relevant information, particularly from the olfactory system. While GnRH and steroid receptors are present in olfactory processing regions across vertebrates, little is known about whether their expression levels change with internal physiological state or external social cues. We used qRT-PCR to measure mRNA levels of two GnRH receptors (GnRH-R1, GnRH-R2), five sex steroid receptors (estrogen receptors: ERalpha, ERbetaa, ERbetab; androgen receptors: ARalpha, ARbeta), and aromatase in the olfactory bulb of the highly social African cichlid fish Astatotilapia burtoni. We asked whether these receptor levels changed with reproductive condition in females, or with social status, which regulates reproductive capacity in males. Our results reveal that mRNA levels of multiple sex steroid, GnRH receptor subtypes, and aromatase in the olfactory bulb vary with sex, social status in males, and reproductive condition in females, which highlights the potential importance of changing receptor levels in fine-tuning the olfactory system during the reproductive cycle. Further, steroid receptor mRNA levels were positively correlated with circulating steroid levels in males, but negatively correlated in females, suggesting different regulatory control between sexes. These results provide support for the hypothesis that the first-order olfactory relay station is a substrate for both GnRH and sex steroid modulation, and suggest that changes in receptor levels could be an important mechanism for regulating reproductive, social, and seasonal plasticity in olfactory perception observed across vertebrates., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Objectives: Receptors for luteinizing hormone-releasing hormone (LHRH) can be utilized for targeted chemotherapy of cytotoxic LHRH analogs. The compound AEZS-108 (previously AN-152) consists of [D-Lys⁶]LHRH linked to doxorubicin. The objectives of this first study in humans with AESZ-108 were to determine the maximum tolerated dose and to characterize the dose-limiting toxicity, pharmacokinetics, preliminary efficacy, and hormonal effects., Methods: The study included 17 women with histologically confirmed epithelial cancer of the ovary, endometrium, or breast that was metastatic or unresectable and for which standard curative or palliative measures could not be used or were no longer effective or tolerated. In each patient, immunohistochemistry of primary tumor or metastatic lesion confirmed that the tumors expressed LHRH receptors., Results: One patient each received intravenous doses of 10, 20, 40, or 80 mg/m² of AEZS-108, six received 160 mg/m² and seven 267 mg/m² at 3 week intervals. Dose-limiting leukopenia and neutropenia were observed at the highest dose. A total of 6 patients, 3 patients each in both upper dose groups, showed responses to AEZS-108. The half-life of AESZ-108 was estimated to be about 2h., Conclusions: The maximum tolerated dose of AESZ-108 in the absence of supportive medication is 267 mg/m² and this dose is recommended as starting dose for therapeutic Phase II studies., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: We recently demonstrated the effect of gonadotrophin-releasing hormone agonist (GnRHa) on tissue inflammation, angiogenesis and apoptosis in endometriosis, adenomyosis and uterine myoma. Here, we investigated expression of GnRH receptors (GnRHRs) and effect of GnRHa on the proliferation of cells derived from endometria and pathological lesions of women with these reproductive diseases., Methods: Biopsy specimens were collected from lesions and corresponding endometria of 35 women with pelvic endometriosis, 45 women with ovarian endometrioma, 35 women with adenomyosis and 56 women with uterine myoma during laparoscopy or laparotomy. The gene and protein expressions of GnRHR in eutopic/ectopic cells and tissues were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. The immunoreactivity of GnRHR in tissue was analysed by quantitative-histogram (Q-H) scores. The exogenous effect of GnRHa on cell proliferation was examined by 5-bromo-2-deoxyuridine incorporation assay. The Ki-67-immunoreactive cell proliferation index was analysed in biopsy specimens derived from GnRHa-treated and -non-treated women., Results: Types I and II GnRHRs mRNA and proteins were expressed in eutopic endometria and pathological lesions derived from women with endometriosis, adenomyosis and uterine myoma. GnRHR expression was the highest in the menstrual phase when compared with other phases of the menstrual cycle. Higher Q-H scores of GnRHR immunoreaction were found in blood-filled opaque red lesions than in other peritoneal lesions. Exogenous treatment with GnRHa significantly suppressed the proliferation of cells derived from respective endometria and pathological lesions when compared with GnRHa-non-treated cells., Conclusions: Local tissue expression of GnRHR was detected in endometriosis, adenomyosis and uterine myoma. In addition to a hypo-estrogenic effect, a direct anti-proliferative effect of GnRHa may be involved in the regression of these reproductive diseases with consequent remission of clinical symptoms.

Studies indicated that many tissues could express FSH. New functions of FSH have been recognized beyond reproduction regulation. However, no report has been made about the expression and function of FSH in rat spinal cord. Double-labeled immunofluorescence stain and in situ hybridization were used to study the co-localization of FSH with its receptor and co-localization of FSH with GnRH receptor in rat spinal cord. Spinal cord ischemia injury models were built, TUNEL stain and Fas immunostaining were made to observe the anti-apoptotic effects of FSH to neurons induced by spinal cord ischemia injury. The results found that some neurons and glias of rat spinal cord showed both FSH immunoreactivity and FSH mRNA positive signals; not only FSH and its receptor but also FSH and GnRH receptor co-located in cells of both gray matter and white matter; treatment with certain concentration of FSH before ischemia-reperfusion injury, less TUNEL positive cells and Fas positive cells were found in motor neurons of ventral gray matter in FSH experiment group than that in control group. These suggested that rat spinal cord could express FSH, it is also a target organ of FSH; FSH might exert functions through its receptor by paracrine or autocrine effects; GnRH in spinal cord might regulate FSH positive neurons through GnRH receptor; FSH might inhibit ischemia induced neuron apoptosis by down-regulating Fas expression in spinal cord.

Chronic exposure to ethanol suppresses the male reproductive activity which is primarily involved in the release of hypothalamic gonadotropin-releasing hormone (GnRH). The testicular GnRH and GnRH receptors (GnRH-R) are found in seminiferous tubules, which are predicted to act as a local regulator of spermatogenesis, although the function is not well known. In this study, we investigated the chronic ethanol effect on GnRH mRNA expression in hypothalamus and testis using in situ hybridization and RNase protection assay (RPA). The effect of ethanol on expressional changes of GnRH and GnRH-R mRNA was observed in adult and pubertal rats according to age and time from 2 weeks (short term) and 4 weeks (long term) periods. The results showed that GnRH mRNA expression in adult and pubertal rats was dramatically decreased in the testis while no significant change was observed in hypothalamus after both short and long term exposure to ethanol. The pubertal rats showed decrease in testicular GnRH and GnRH-R mRNA expression, whereas GnRH mRNA was increased significantly, while GnRH-R mRNA was further decreased after long term exposure in adults. This study suggested that chronic ethanol administration is more effective to testicular GnRH and GnRH-R mRNA expression than hypothalamus and causes a negative effect on the spermatogenesis process. Furthermore, our finding suggests that the deteriorative effects of ethanol on gonadal activity are more lethal in puberty than adults., (2010 Elsevier Ireland Ltd. All rights reserved.)

Background/aims: Hypothalamic hamartomas are the most common identifiable cause of central precocious puberty (CPP). Hamartoma characteristics proposed to be associated with CPP include specific anatomic features and expression of molecules such as gonadotropin-releasing hormone (GnRH), transforming growth factor alpha (TGFalpha), and GRM1A, which encodes the type 1 metabotropic glutamate receptor alpha isoform. We sought to determine whether hamartomas that cause CPP could be distinguished by anatomic features, expression of these molecules, or expression of KISS1, whose products signal through the receptor GPR54 to stimulate GnRH release., Methods: Clinical records and radiologic images were reviewed for 18 patients who underwent hamartoma resection for intractable seizures; 7 had precocious puberty. Resected tissue was examined for expression of GnRH, GnRH receptor (GnRHR), TGFalpha, KISS1, GPR54, and GRM1A., Results: Hypothalamic hamartomas associated with CPP were more likely to contact the infundibulum or tuber cinereum and were larger than hamartomas not associated with CPP. GnRH, TGFalpha, and GnRHR were expressed by all hamartomas studied. Expression of KISS1, GPR54, and GRM1A did not differ significantly between hamartomas associated and not associated with CPP., Conclusion: Anatomic features rather than expression patterns of candidate molecules distinguish hypothalamic hamartomas that are associated with CPP from those that are not., (Copyright 2010 S. Karger AG, Basel.)

The presence of four different isoforms of luteinizing hormone-releasing hormones (LHRH) and one LHRH receptor (LHRH-R) has been reported in vertebrates. In the human genome only LHRH-I and LHRH-II genes have been identified. The human LHRH-I gene is composed of four exons separated by three introns. Three LHRH receptor or receptor-like genes have been demonstrated. The well-established type-I LHRH receptor (LHRH-R-I) gene is composed of three exons separated by two introns. In this study we investigated the expression of transcript forms of LHRH-R-I in human benign prostatic hyperplasia (BPH) with reverse transcriptase-polymerase chain reaction (RT-PCR) using gene specific primers. Thirty-five human BPH specimens were obtained at surgery. Normal human pituitaries collected at autopsy served as control. RNA extraction and RT-PCR with gene-specific primers for LHRH-R-I forward (F1)/reverse (R1), LHRH-R-I F2/R3, LHRH-R-I F1'/R2' were carried out to determine the mRNA expression for LHRH-R-I transcript forms. The expected PCR products amplified with gene specific primers were LHRH-R-I F1/R1 with 319 bp, LHRH-R-I F2/R3 with 309 bp and LHRH-R-I F1'/R2' with 219 bp. PCR products for LHRH-R-I F1/R1 were detected in 21 (60%) and for LHRH-R-I F2/R3 in 5 of 35 (14%) BPH samples. No PCR products for LHRH-R-I F1'/R2' were found. In conclusion, we detected mRNA for LHRH-R-I in human BPH specimens. Our results suggest that LHRH-R-I gene may have more than two splice variants or uncharacterised transcript forms of LHRH-R-I. Our findings support the merit of further investigation of the expression of LHRH-R-I and its transcript forms in human BPH.

Brain sex steroids are derived from both peripheral (primarily gonadal) and local (neurosteroids) sources and are crucial for neurogenesis, neural differentiation and neural function. The mechanism(s) regulating the production of neurosteroids is not understood. To determine whether hypothalamic-pituitary-gonadal axis components previously detected in the extra-hypothalamic brain comprise a feedback loop to regulate neuro-sex steroid (NSS) production, we assessed dynamic changes in expression patterns of steroidogenic acute regulatory (StAR) protein, a key regulator of steroidogenesis, and key hypothalamic-pituitary-gonadal endocrine receptors, by modulating peripheral sex hormone levels in female mice. Ovariectomy (OVX; high serum gonadotropins, low serum sex steroids) had a differential effect on StAR protein levels in the extrahypothalamic brain; increasing the 30- and 32-kDa variants but decreasing the 37-kDa variant and is indicative of cholesterol transport into mitochondria for steroidogenesis. Treatment of OVX animals with E(2), P(4), or E(2) + P(4) for 3 days, which decreases OVX-induced increases in GnRH/gonadotropin production, reversed this pattern. Suppression of gonadotropin levels in OVX mice using the GnRH agonist leuprolide acetate inhibited the processing of the 37-kDa StAR protein into the 30-kDa StAR protein, confirming that the differential processing of brain StAR protein is regulated by gonadotropins. OVX dramatically suppressed extra-hypothalamic brain gonadotropin-releasing hormone 1 receptor expression, and was further suppressed in E(2)- or P(4)-treated OVX mice. Together, these data indicate the existence of endocrine and autocrine/paracrine feedback loops that regulate NSS synthesis. Further delineation of these feedback loops that regulate NSS production will aid in developing therapies to maintain brain sex steroid levels and cognition.

The GnRH receptor (GnRHR) responds to pulsatile GnRH signals to coordinate pituitary gonadotropin synthesis and secretion. Previously, a 1.2-kb fragment of the 5'-flanking region isolated from the mouse GnRHR gene was shown to target expression to pituitary gonadotropes in vivo. The 1.2-kb gene promoter fused to the simian virus 40 large T antigen (TAg) was used to generate transgenic mice that form gonadotrope-derived pituitary tumors at 4-5 months of age. Transgenic female mice have hypogonadotropic hypogonadism, infantile gonads, and are infertile throughout their life span, whereas males remain reproductively intact until their tumors become large. We hypothesized that the targeted TAg expression causes a sex-specific disruption of the reproductive axis at the level of the pituitary gland. To test this hypothesis, we characterized the pituitary gonadotropin beta-subunit and TAg expression patterns, and measured plasma gonadotropin and gonadal steroid levels in female and male mice before and after pituitary tumor development. TAg expression was observed in transgenic females and males 15 d of age, before tumor development. Interestingly, and in contrast to the transgenic males, pituitary LH beta and FSH beta subunit protein levels, and plasma LH and FSH levels, were reduced in transgenic females. Reproductive organs in transgenic female mice remained underdeveloped but were normal in transgenic males. We conclude that the expression of the TAg transgene driven by the GnRHR gene promoter results in female-specific infertility due to disruption of gonadotropin production and secretion even before tumor development.

Malignant glioblastoma is one of the highest proliferative and invasive tumors within the central nervous system (CNS); the therapeutical options for this disease are still very poor. Receptors for gonadotropin-releasing hormone (GnRH) have been reported to be present in glioblastoma tissues. This study aimed to determine the role of these receptors in the control of glioma growth. In two human glioblastoma cell lines, U87MG and U373, we demonstrated the expression of GnRH receptors, both at mRNA and protein levels. We also found that GnRH receptor is expressed in glioblastoma tissues from tumor patients as shown by Western blotting. In U87MG and U373 cell lines, we found the expression of mRNA for GnRH, indicating the presence of an autocrine GnRH-based system in these cell lines. Treatment of the two cell lines with a GnRH agonist resulted in a significant decrease of cell proliferation. Moreover, the GnRH agonist significantly counteracted the forskolin-induced increase of intracellular cAMP levels in these cells. These findings suggest that the GnRH receptor might represent a molecular target for an endocrine therapeutical approach against gliomas.

The effect of prolonged, intermittent infusion of GABA(A) receptor agonist (muscimol) or GABA(A) receptor antagonist (bicuculline) into the third cerebral ventricle on the expression of GnRH gene and GnRH-R gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland was examined in follicular-phase ewes by real-time PCR. The activation or inhibition of GABA(A) receptors in the hypothalamus decreased or increased the expression of GnRH and GnRH-R genes and LH secretion, respectively. The present results indicate that the GABAergic system in the hypothalamus of follicular-phase ewes may suppress, via hypothalamic GABA(A) receptors, the expression of GnRH and GnRH-R genes in this structure. The decrease or increase of GnRH-R mRNA in the anterior pituitary gland and LH secretion in the muscimol- or bicuculline-treated ewes, respectively, is probably a consequence of parallel changes in the release of GnRH from the hypothalamus activating GnRH-R gene expression. It is suggested that GABA acting through the GABA(A) receptor mechanism on the expression of GnRH gene and GnRH-R gene in the hypothalamus may be involved in two processes: the biosynthesis of GnRH and the release of this neurohormone in the hypothalamus.

Several studies have previously reported the expression of the gonadotropin-releasing hormone receptor (GnRHr) in cases of endometrial cancer. However, the relationship between GnRHr expression and a variety of clinicopathologic parameters remains unclear. This study was conducted with 141 endometrial cancer patients, all of whom had undergone operations between 1993 and 2002. Paraffin-embedded tissue blocks were sectioned and immunostained with monoclonal anti-GnRHr antibody. Clinicopathologic variables were also evaluated, with 10% cutoff values for GnRHr positivity. Seventy specimens (49.6%) stained as GnRHr-positive. Mean parity was higher in the patients with GnRHr-positive tumors than those with GnRHr-negative tumors (2.50+/-1.92 versus 1.82+/-1.37, P=0.016). Body mass indices were also higher in the patients with GnRHr-positive tumors (26.6+/-4.6 versus 24.7+/-4.2, P=0.010). However, GnRHr positivity was not determined to be statistically significantly associated with any other clinicopathologic characteristics, including age, menopausal status, histotype, disease stage, tumor differentiation, lymph node metastasis, and myometrial invasion. The results of this study, although they may require further investigation, suggested that obese and multiparous women with endometrial cancer might be greatly influenced by endogenous gonadotropin-releasing hormone and/or exogenous gonadotropin-releasing hormone analogs.

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

The differential expression of mRNA and protein of GnRH I, II and their receptors (RI and RII) in the monkey corpus luteum (CL) were measured during different stages of the luteal phase of the menstrual cycle as an initial step towards considering the role and regulation of GnRH (I and II) system during luteinization and luteolysis in primates. RT-PCR confirmed the sequence identity of PCR products and real time PCR quantified specific mRNA expressions. Proteins were localized by immunohistochemistry (IHC). Changes in mRNA expression patterns of GnRH I and II (increased) and GnRH RII (decreased) were maximal at mid-late to late stages, that is, at CL regression, where as GnRH RI was low during the entire luteal phase. However, RT-PCR and IHC studies confirmed the presence of GnRH RI at both mRNA and protein levels, respectively. IHC results showed the presence of GnRH I, II and their receptors in steroidogenic cells (granulose-luteal cells and thecal-luteal cells) across the luteal phase. Hence, GnRH I and II systems may have a role on both luteinization (from early to mid stages of CL) and luteolysis (from mid-late to very-late stages of CL). These novel findings suggest that monkey luteal GnRH system may have a role in fertility regulation in paracrine and/or autocrine manner.

This paper describes the development and validation of a PCR array for studying chemical-induced effects on gene expression of selected endocrine pathways along the hypothalamic-pituitary-gonadal (HPG) axis of the small, oviparous fish, the Japanese medaka (Oryzias latipes). The Japanese medaka HPG-PCR array combines the quantitative performance of SYBR Green-based real-time PCR with the multiple gene profiling capabilities of a microarray to examine expression profiles of 36 genes associated with endocrine pathways in brain, liver and gonad. The performance of the Japanese medaka HPG-PCR array was evaluated by examining effects of two model compounds, the synthetic estrogen, 17alpha-ethinylestradiol (EE2) and the anabolic androgen, 17beta-trenbolone (TRB) on the HPG axis of the Japanese medaka. Four-month-old medaka was exposed to three concentrations of EE2 (5, 50, 500 ng/L) or TRB (50, 500, 5000 ng/L) for 7d in a static renewal exposure system. A pathway-based approach was implemented to analyze and visualize concentration-dependent mRNA expression in the HPG axis of Japanese medaka. The compensatory response to EE2 exposure included the down-regulation of male brain GnRH RI and testicular CYP17. The down-regulation of AR-alpha expression in brain of EE2-exposed males was associated with suppression of male sexual behavior. Compensatory responses to TRB in the female HPG axis included up-regulation of brain GnRH RII and ovary steroidogenic CYP19A. Overall, the results suggested that the Japanese medaka HPG-PCR array has potential not only as a screening tool of potential endocrine-disrupting chemicals but also in elucidating mechanisms of action.

In the present paper the role of GnRH in the ultrashort loop of the negative feedback action on GnRH secretion was evaluated on the molecular level by the Real-time PCR technique. Specifically, the effect of GnRH infused into the third cerebral ventricle on the expression of GnRH and GnRH receptor (GnRH-R) genes was analyzed in the hypothalamic-pituitary unit of anestrous ewes. GnRH did not significantly affect GnRH mRNA levels in the preoptic/anterior hypothalamic area but drastically increased its level in the ventromedial hypothalamus. In addition, GnRH infusion augmented GnRH-R mRNA level in the entire hypothalamus. In the GnRH-treated animals, anterior pituitary GnRH-R mRNA level and plasma LH concentration were also elevated. The changes in GnRH mRNA and GnRH-R mRNA levels in the hypothalamus in response to treatment with GnRH suggest that GnRH acts differently on the stability of these transcripts. On the basis of presented results it seems that GnRH may affect GnRH and GnRH-R biosynthesis and, consequently, GnRH/LH release.

Background: The majority of human breast cancers and in addition most breast-cancer cell lines express gonadotropin-releasing hormone (GnRH) receptors. Their proliferation and in addition their bone-directed invasion is time- and dose-dependently reduced by GnRH. Osteolytic metastases are characteristic for breast cancer-derived metastasis. Since the osteolytic activity depends on the receptor activator of nuclear factor-kappaB (NFkappaB) ligand (RANKL)/osteoprotegerin (OPG) ratio, we analyzed RANKL and OPG expression in different breast-cancer cell lines., Methods: Different human breast-cancer cell lines were tested for expression of GnRH receptor, OPG and RANKL. Using a co-culture system of breast-cancer cell lines and human primary osteoblasts (hOB), we analyzed the expression of OPG and RANKL in the GnRH receptor-positive breast-cancer cell line HCC70 co-cultured with or without hOB. In addition, we assessed the effects of GnRH analog treatment on OPG and RANKL mRNA and protein levels., Results: All tested breast-cancer cell lines were GnRH receptor-positive. The majority of these cell lines expressed OPG but not RANKL. The HCC70 breast-cancer cell line derived from an invasive ductal carcinoma with metastases was positive for both OPG and RANKL. The expression of RANKL by HCC70 cells was increased when co-cultured with hOB. Treatment with GnRH analogs reduced the expression of RANKL by HCC70 cells co-cultured with hOB. No effects were observed on breast cancer OPG expression., Conclusions: These data show that the majority of human breast-cancer cell lines express OPG but not RANKL. The HCC70 breast-cancer cell line is RANKL-positive. Co-culture of HCC70 breast cancer cells with hOB increases RANKL expression. Activation of tumor GnRH receptors reduces RANKL expression. These experiments demonstrate that HCC70 breast cancer cells are able to activate osteoclasts directly via RANKL. The interaction between HCC70 breast cancer cells and osteoblasts induces osteoclastogenesis through an increase of RANKL expression. GnRH seems to play an important role by modulating the RANKL expression in HCC70 breast cancer cells.

A recent study from our laboratory has shown cellular and ultrastructural distribution of the gonadotropin-releasing hormone (GnRH) and the relative expression of its mRNA in the rat oviduct during the postimplantation period of pregnancy (days 7, 9, 16, and 20). To determine the possible autocrine/paracrine involvement of the oviductal GnRH during pregnancy in rats, the present investigation aims at the study of the relative expression of GnRH receptor (GnRHR) mRNA by real-time PCR followed by immunolocalization of the peptide in the oviduct during pregnancy. Semiquantitative analysis of the oviductal GnRHR expression by Western blot was done by densitometry of the signal intensity. Our results indicate the expression of GnRHR mRNA in the rat oviduct throughout the postimplantation period of pregnancy with no significant difference in expression between the selected time points. Immunoreactive GnRHR peptide was localized predominantly in the cytoplasm of the luminal epithelial cells, with less expression in the cytoplasm of the stromal cells and the smooth muscles throughout the oviduct. Signal intensity of GnRHR was significantly lower during day 16 when compared to days 7 and 20. These results, for the first time, support the transcription of GnRHR mRNA and its translation to protein in the rat oviduct throughout the postimplantation period of pregnancy. The lower protein content of GnRHR by day 16 may be indicative of ligand-induced downregulation of the receptor expression. The present investigation thus strengthens our previously postulated hypothesis regarding the receptor-mediated autocrine/paracrine role of oviductal GnRH during pregnancy in rats.

Objective: To determine the presence or absence of a second form of GnRH (GnRH2) and corresponding receptor (GnRHR2) in human uterine myometrium and leiomyomata., Design: Evaluation of human leiomyoma and patient-matched myometrium of differential mRNA and protein expression of GnRH2 and GnRHR2., Setting: University hospital., Patient(s): Eight women undergoing medically indicated hysterectomy for symptomatic fibroids., Intervention(s): Microarray analysis, reverse-transcriptase polymerase chain reaction (RT-PCR), real-time RT-PCR, and immunohistochemistry., Main Outcome Measure(s): Expression of mRNA and protein in leiomyoma and patient-matched myometrium., Result(s): Microarray analysis demonstrated expression, and we confirmed the findings by RT-PCR. Real-time RT-PCR demonstrated equivalent expression of the genes in leiomyoma compared with patient-matched myometrium (0.99-fold for GnRH2 and 1.28-fold for GnRHR2). Immunohistochemistry confirmed the expression of GnRH2 protein in both leiomyoma and myometrium., Conclusion(s): A second form of GnRH and corresponding receptor exists in the fibroid and myometrium. We speculate that an autocrine loop exists. Our findings provide further evidence that GnRH agonists may interact directly with GnRH receptors present in uterine fibroids.

Gonadotropin-releasing hormone (GnRH) I and II are hypothalamic decapeptides with pivotal roles in the development of reproductive competence and regulation of reproductive events. In this study, transcriptional regulation of the human GnRH II gene was investigated. By scanning mutation analysis coupled with transient promoter assays, the motif at -641/-636 (CATGCC, designated GII-Sil) was identified as a repressor element. Mutation of this motif led to full restoration of promoter activity in TE671 medulloblastoma and JEG-3 placenta choriocarcinoma cells. Supershift and chromatin immunoprecipitation assays showed in vitro and in vivo binding of NF-kappaB subunit p65 and the retinoic acid receptors, RARalpha and RXRalpha, to the promoter sequences. Over-expression of these protein factors indicated that p65 is a potent repressor, and the RARalpha/RXRalpha heterodimer is involved in the differential regulation of the GnRH II gene in neuronal and placental cells. This was confirmed by quantitative real-time PCR. Treatment of cells with the RARalpha/RXRalpha ligands, all-trans retinoic acid and 9-cis-retinoic acid, reduced and increased GnRH II gene expression in TE671 and JEG-3 cells, respectively. Taken together, these data demonstrate the differential roles of NF-kappaB p65 and RARalpha/RXRalpha, interacting with the same sequence in the promoter of the human GnRH II gene to influence gene expression in a cell-specific manner.