Gonadotropin-Releasing Hormone Receptor Expression in Human Spinal Cord.

The expression of the gonadotrophin-releasing hormone receptor expression on pituitary gonadotrophs in humans is well characterized. In nervous system they have also been found in hippocampi and cerebral cortex. However, gonadotrophin-releasing hormone receptor expression in human spinal cord has not been reported. This study was to analyze the gonadotrophin-releasing hormone receptor expression in human spinal cord by immunohistochemistry, mRNAs by reverse transcriptase polymerase chain reaction, cDNA cloning and Western blot. The results show immunoreactive material to gonadotrophin-releasing hormone receptor in motoneurons of the spinal cord. Further, the study revealed that spinal cord expressed the gonadotrophin-releasing hormone receptor mRNA. The amplicon sequence corresponds to 100% of identity to GenBank. In Western blot, a band of 37 kDa were found in extracts of spinal cord and placenta as a control. In conclusion, human spinal cord expresses gonadotrophin-releasing hormone receptor analyzed through immunohistochemistry, the expression of its mRNA, cloning its cDNA and Western blot analysis. The presence of gonadotrophin-releasing hormone receptor in the spinal cord suggests the possibility of an extrapituitary functional role independent of reproductive system.

Mammary gland-specific regulation of GNRH and GNRH-receptor gene expression is likely part of a local autoregulatory system in female vizcachas (Rodentia: Chinchillidae).

In addition to key mammotrophic hormones such as the pituitary prolactin (PRL) and the ovarian steroids progesterone and estradiol, there are local factors that modulate the tissue dynamics of the mammary glands during pregnancy and lactation. By immunohistochemistry and RT-PCR, we found local transcription and translation of gonadotropin-releasing hormone (GNRH), GNRH receptor (GNRHR), PRL and PRL receptor (PRLR) in mammary glands of adult vizcachas during pregnancy and lactation. Both GNRH and GNRHR showed a lag between protein expression and gene transcription throughout the gestational period: while the highest transcription levels of these genes were recorded at early-pregnancy, the epithelial immunoexpressions of both showed their maximum during lactation. RIA results corroborated the presence of GNRH in mammary glands at all the analyzed stages and confirmed the maximum amount of this peptide in the lactating group. Significant amounts of GNRH were detected in milk samples as well. Conversely, PRL and PRLR shared similar protein and gene expression profiles, all exhibiting maximum values during lactation. GNRH peptide content in mammary glands of females with sulpiride-induced hyperprolactinemia (HP) was significantly lower than that of control females (CT). Although PRL mRNA levels remained unchanged, there was a marked increase in theα-lactalbumin (LALBA) transcription in mammary glands of HP- vs CT-females. These results suggest that after targeting mammary glands, PRL stimulates the expression of milk protein genes, but also, tempers the local expression of GNRH. Mammary gland-explantssupplemented with a GNRH analogue (GN-explants) had no differences in terms of PRLR orLALBA transcription levels compared to CT-explants, so the mammary PRLR signaling would not appear to be modulated by GNRH. Yet, mRNA expression levels of both GNRH and the GNRHR-downstream factor, EGR1, were significantly higher in GN-explants compared to that of CT which would point to a GNRH-positive feedback mechanism. In summary, the local coupled expression of GNRH, GNRHR and EGR1 in the mammary gland throughout pregnancy of vizcachas, the PRL-dependent mammary GNRH secretion as well as the GNRH positive feedback on its own transcription suggest an autocrine-paracrine regulatory mechanism and propose an active role for GNRH in mammary gland tissue remodeling.

Variants in the Kisspeptin-GnRH Pathway Modulate the Hormonal Profile and Reproductive Outcomes.

Kisspeptin has been identified as a key regulatory protein in the release of gonadotropin-releasing hormone (GnRH), which subsequently increases gonadotropin secretion during puberty to establish reproductive function and regulate the hypothalamic-pituitary-gonadal axis. The effects of variants in the KISS1, KISS1R, and GNRHR genes and their possible association with assisted reproduction outcomes remain to be elucidated. In this study, we used next-generation sequencing to investigate the associations of the genetic diversity at the candidate loci for KISS1, KISS1R, and GNRHR with the hormonal profiles and reproductive outcomes in 86 women who underwent in vitro fertilization treatments. Variants in the KISS1 and KISS1R genes were associated with luteinizing hormone (rs35431622:T>C), anti-Mullerian hormone (rs71745629delT), follicle-stimulating hormone (rs73507529:C>A), and estradiol (rs73507527:G>A, rs350130:A>G, and rs73507529:C>A) levels, as well as with reproductive outcomes such as the number of oocytes retrieved (s35431622:T>C), metaphasis II oocytes (rs35431622:T>C), and embryos (rs1132506:G>C). Additionally, variants in the GNRHR UTR3' (rs1038426:C>A, rs12508464:A>C, rs13150734:C>A, rs17635850:A>G, rs35683646:G>A, rs35610027:C>G, rs35845954:T>C, rs17635749:C>T, and rs7666201:C>T) were associated with low prolactin levels. A conjoint analysis of clinical, hormonal, and genetic variables using a generalized linear model identified two variants of the KISS1 gene (rs71745629delT and rs1132506:G>C) that were significantly associated with hormonal variations and reproductive outcomes. The findings suggest that variants in KISS1, KISS1R, and GNRHR genes can modulate hormone levels and reproductive outcomes.

Cancer stem cell and mesenchymal cell cooperative actions in metastasis progression and hormone resistance in prostate cancer: Potential role of androgen and gonadotropin­releasing hormone receptors (Review).

Prostate cancer (PCa) is the leading cause of male cancer­associated mortality worldwide. Mortality is associated with metastasis and hormone resistance. Cellular, genetic and molecular mechanisms underlying metastatic progression and hormone resistance are poorly understood. Studies have investigated the local effects of gonadotropin­releasing hormone (GnRH) analogs (used for androgen deprivation treatments) and the presence of the GnRH receptor (GnRH­R) on PCa cells. Furthermore, cell subpopulations with stem­like properties, or cancer stem cells, have been isolated and characterized using a cell culture system derived from explants of human prostate tumors. In addition, the development of preclinical orthotopic models of human PCa in a nonobese diabetic/severe combined immunodeficiency mouse model of compromised immunity has enabled the establishment of a reproducible system of metastatic progression in vivo. There is increasing evidence that metastasis is a complex process involving the cooperative actions of different cancer cell subpopulations, in which cancer stem­like cells would be responsible for the final step of colonizing premetastatic niches. It has been hypothesized that PCa cells with stemness and mesenchymal signatures act cooperatively in metastatic progression and the inhibition of stemness genes, and that overexpression of androgen receptor (AR) and GnRH­R decreases the rate the metastasis and sensitizes tumors to hormone therapy. The aim of the present review is to analyze the evidence regarding this cooperative process and the possible influence of stem­like cell phenotypes, AR and GnRH­R in metastatic progression and hormone resistance. These aspects may represent an important contribution in the understanding of the mechanisms underlying metastasis and hormone resistance in PCa, and potential routes to blocking these processes, enabling the development of novel therapies that would be particularly relevant for patients with metastatic and castration­resistant PCa.

Enteroendocrine Connections in Congenital Isolated GH Deficiency Due to a GHRH Receptor Gene Mutation.

CONTEXT: GH and IGF-1 are crucial for attainment of normal body size and regulation of food intake, nutrient storage, and insulin sensitivity. Enteroendocrine connections exist between the GH-IGF-1 axis and insulin, ghrelin, and glucagon-like peptide 1 (GLP-1). The status of these connections in GH deficiency (GHD) is unknown. OBJECTIVE: To study the enteroendocrine connections before and after a standard meal test in a homogeneous population of adults with congenital untreated isolated GHD (IGHD) due to a mutation in the GHRH receptor gene. DESIGN: In a cross-sectional study of 20 individuals with IGHD and 20 control subjects, we measured glucose, insulin, ghrelin, and GLP-1 before and 30, 60, 120, and 180 minutes after a standardized test meal. Homeostasis model assessment index of insulin resistance (HOMA-IR) and homeostasis model assessment (HOMA)-ß were calculated. Participants scored feelings of hunger, fullness, and prospective food consumption on a visual analog scale. MAIN OUTCOME MEASURES: Area under the curve (AUC) values of glucose, insulin, ghrelin, GLP-1, hunger, fullness, and prospective food consumption. RESULTS: Fasting HOMA-IR and HOMA-ß were lower in individuals with IGHD than in control subjects (P = 0.002 and P = 0.023, respectively). AUC was higher for hunger (P < 0.0001), glucose (P = 0.0157), ghrelin (P < 0.0001), and GLP-1 (P < 0.0001) and smaller for fullness (P < 0.0001) in individuals with IGHD compared with control subjects. There was no difference in AUC for prospective food consumption or insulin. CONCLUSIONS: Untreated IGHD is associated with increased GLP-1 secretion and reduced postprandial ghrelin and hunger attenuation in response to a mixed meal. These enteroendocrine connections can result in a favorable outcome in terms of environmental adaptation and guaranteeing appropriate food intake and can confer metabolic benefits.

A novel GNRHR gene mutation causing congenital hypogonadotrophic hypogonadism in a Brazilian kindred.

Congenital hypogonadotrophic hypogonadism (CHH) is a challenging inherited endocrine disorder characterised by absent or incomplete pubertal development and infertility as a result of the low action/secretion of the hypothalamic gonadotrophin-releasing hormone (GnRH). Given a growing list of gene mutations accounting for CHH, the application of massively parallel sequencing comprises an excellent molecular diagnostic approach because it enables the simultaneous evaluation of many genes. The present study proposes the use of whole exome sequencing (WES) to identify causative and modifying mutations based on a phenotype-genotype CHH analysis using an in-house exome pipeline. Based on 44 known genes related to CHH in humans, we were able to identify a novel homozygous gonadotrophin-releasing hormone receptor (GNRHR) p.Thr269Met mutant, which segregates with the CHH kindred and was predicted to be deleterious by in silico analysis. A functional study measuring intracellular inositol phosphate (IP) when stimulated with GnRH on COS-7 cells confirmed that the p.Thr269Met GnRHR mutant performed greatly diminished IP accumulation relative to the transfected wild-type GnRHR. Additionally, the proband carries three heterozygous variants in CCDC141 and one homozygous in SEMA3A gene, although their effects with respect to modifying the phenotype are uncertain. Because they do not segregate with reproductive phenotype in family members, we advocate they do not contribute to CHH oligogenicity. WES proved to be useful for CHH molecular diagnosis and reinforced its benefit with respect to identifying heterogeneous genetic disorders. Our findings expand the GnRHR mutation spectrum and phenotype-genotype correlation in CHH.

Altered sleep patterns in patients with non-functional GHRH receptor.

OBJECTIVES: GH-releasing hormone (GHRH) exerts hypnotic actions increasing the non-rapid eye movement (NREM) sleep. Conversely, GH stimulates the REM sleep. GH deficiency (GHD) often leads to sleep problems, daytime fatigue and reduced quality of life (QoL). GHD may be due to lack of hypothalamic GHRH or destruction of somatotroph cells. We have described a cohort with isolated GHD (IGHD) due to GHRH resistance caused by a homozygous null mutation (c.57 + 1G > A) in the GHRH receptor gene. They have normal QoL and no obvious complaints of chronic tiredness. The aim of this study was to determine the sleep quality in these subjects. METHODS: A cross-sectional study was carried out in 21 adult IGHD subjects, and 21 age- and gender-matched controls. Objective sleep assessment included polygraphic records of the awake, stages NREM [N1 (drowsiness), N2 and N3 (already sleeping)] and REM (R). Subjective evaluation included the Pittsburgh Sleep Quality Index, the Insomnia Severity Index and the Epworth Sleepiness Scale. RESULTS: IGHD subjects showed a reduction in sleep efficiency (P = 0.007), total sleep time (P = 0.028), duration of N2 and R in minutes (P = 0.026 and P = 0.046 respectively), but had increased duration and percentage of N1 stage (P = 0.029 and P = 0.022 respectively), wake (P = 0.007) and wake-time after sleep onset (P = 0.017). There was no difference in N3 or in sleep quality questionnaire scores. CONCLUSION: Patients with IGHD due to GHRH resistance exhibit objective reduction in the sleep quality, with changes in NREM and REM sleep, with no detectable subjective consequences. GHRH resistance seems to have a preponderant role over GHD in the sleep quality of these subjects.

Characterization of Gnrh/Gnih elements in the olfacto-retinal system and ovary during zebrafish ovarian maturation.

Gonadotropin releasing hormone (GnRH) is one of the key players of brain-pituitary-gonad axis, exerting overall control over vertebrate reproduction. In zebrafish, two variants were characterized and named as Gnrh2 and Gnrh3. In this species, Gnrh3, the hypohysiotropic form, is expressed by neurons of the olfactory-retinal system, where it is related with food detection, intra/interspecific recognition, visual acuity and retinal processing modulation. Previous studies have reported the presence of Gnrh receptors in the zebrafish retina, but not yet in the zebrafish olfactory epithelium. The current study analyzed the presence of gnrh2 and gnrh3, their receptors (gnrhr 1,2,3 and 4) and gnih (gonadotropin inhibitory hormone) transcripts, as well as the Gnrh3 protein in the olfactory epithelium (OE), olfactory bulb (OB), retina and ovary during zebrafish ovarian maturation. We found an increase of gnrh receptors transcripts in the OE at the final stages of ovarian maturation. In the OE, Gnrh3 protein was detected in the olfactory receptor neurons cilia and in the olfactory nerve fibers. Interestingly, in the OB, we found an inverse expression pattern between gnih and gnrh3. In the retina, gnrhr4 mRNA was found in the nuclei of amacrine, bipolar, and ganglion cells next to Gnrh3 positive fibers. In the ovary, gnrh3, gnrhr2 and gnrhr4 transcripts were found in perinucleolar oocytes, while gnih in oocytes at the cortical alveolus stage. Our results suggested that Gnrh/Gnih elements are involved in the neuromodulation of the sensorial system particularly at the final stages of maturation, playing also a paracrine role in the ovary.

Equine chorionic gonadotropin influence on sheep oocyte in vitro maturation, apoptosis, and follicle-stimulating hormone receptor and luteinizing hormone receptor expression.

We assessed the effects of equine chorionic gonadotropin (eCG) on oocyte in vitro maturation (IVM), apoptosis, and follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), and gonadotropin-releasing hormone receptor (GnRHR) expression and mRNA levels. Cumulus-oocyte complexes (COCs) were recovered from sheep ovaries and pooled in groups, before being cultured in IVM media containing varying eCG concentrations. Maturation and apoptosis rates were then calculated. Expression of FSHR, LHR, and GnRHR mRNA in oocytes was measured using quantitative reverse transcription polymerase chain reaction. Protein levels were ascertained by western blotting. Matured oocytes displayed and released an intact first polar body. Sheep oocyte maturation rates gradually increased as eCG concentration was raised from 0 to 20 µg/mL. Apoptosis rates of eCG-treated oocytes were lower than those of the control group, and were lowest using 20 µg/mL eCG. FSHR, LHR, and GnRHR mRNA expression increased (P < 0.01, P < 0.05, and P < 0.05, respectively, compared to 0 µg/mL eCG) with eCG concentration, being highest following exposure to 20 µg/mL. FSHR and GnRHR protein levels were significantly higher in oocytes administered 20 µg/mL eCG compared with those matured in the absence of eCG. eCG dose positively correlated with FSHR, LHR, and GnRHR mRNA and protein expression. In conclusion, eCG enhances maturation and decreases apoptosis of oocytes undergoing IVM, and heightens FSHR, LHR, and GnRHR expression. Such increased expression may facilitate oocyte IVM. These findings contribute to our understanding of the mechanisms of underlying hormonal control of sheep oocyte IVM, advancing ovine reproductive methods.

Adult exposure to bisphenol A (BPA) in Wistar rats reduces sperm quality with disruption of the hypothalamic-pituitary-testicular axis.

Reproductive physiology involves complex biological processes that can be disrupted by exposure to environmental contaminants. The effects of bisphenol A (BPA) on spermatogenesis and sperm quality is still unclear. The objective of this study was to investigate the reproductive toxicity of BPA at dosages considered to be safe (5 or 25mg BPA/kg/day). We assessed multiple sperm parameters, the relative expression of genes involved in the central regulation of the hypothalamic-pituitary-testicular axis, and the serum concentrations of testosterone, estradiol, LH and FSH. BPA exposure reduced sperm production, reserves and transit time. Significant damage to the acrosomes and the plasma membrane with reduced mitochondrial activity and increased levels of defective spermatozoa may have compromised sperm function and caused faster movement through the epididymis. BPA exposure reduced the serum concentrations of testosterone, LH and FSH and increased the concentration of estradiol. The relative gene expression revealed an increase in gonadotropin releasing hormone receptor (Gnrhr), luteinizing hormone beta (Lhb), follicle stimulating hormone beta (Fshb), estrogen receptor beta (Esr2) and androgen receptor (Ar) transcripts in the pituitary and a reduction in estrogen receptor alpha (Esr1) transcripts in the hypothalamus. In this study, we demonstrated for the first time that adult male exposure to BPA caused a reduction in sperm production and specific functional parameters. The corresponding pattern of gene expression is indicative of an attempt by the pituitary to reestablish normal levels of LH, FSH and testosterone serum concentrations. In conclusion, these data suggest that at dosages previously considered nontoxic to reproductive function, BPA compromises the spermatozoa and disrupts the hypothalamic-pituitary-gonadal axis, causing a state of hypogonadotropic hypogonadism.

Role of gonadotropin-releasing hormone receptor mutations in patients with a wide spectrum of pubertal delay.

OBJECTIVE: To analyze the GNRHR in patients with normosmic isolated hypogonadotropic hypogonadism (IHH) and constitutional delay of growth and puberty (CDGP). DESIGN: Molecular analysis and in vitro experiments correlated with phenotype. SETTING: Academic medical center. PATIENT(S): A total of 110 individuals with normosmic IHH (74 male patients) and 50 with CDGP. INTERVENTION(S): GNRHR coding region was amplified and sequenced. MAIN OUTCOME MEASURE(S): Novel variants were submitted to in vitro analysis. Frequency of mutations and genotype-phenotype correlation were analyzed. Microsatellite markers flanking GNRHR were examined in patients carrying the same mutation to investigate a possible founder effect. RESULT(S): Eleven IHH patients (10%) carried biallelic GNRHR mutations. In vitro analysis of novel variants (p.Y283H and p.V134G) demonstrated complete inactivation. The founder effect study revealed that Brazilian patients carrying the p.R139H mutation shared the same haplotype. Phenotypic spectrum in patients with GNRHR mutations varied from complete GnRH deficiency to partial and reversible IHH, with a relatively good genotype-phenotype correlation. One boy with CDGP was heterozygous for the p.Q106R variant, which was not considered to be pathogenic. CONCLUSION(S): GNRHR mutations are a frequent cause of congenital normosmic IHH and should be the first candidate gene for genetic screening in this condition, especially in autosomal recessive familial cases. The founder effect study suggested that the p.R139H mutation arises from a common ancestor in the Brazilian population. Finally, mutations in GNRHR do not appear to be involved in the pathogenesis of CDGP.

Gonadotropin-releasing hormone receptor activates GTPase RhoA and inhibits cell invasion in the breast cancer cell line MDA-MB-231.

BACKGROUND: Gonadotropin-releasing hormone (GnRH) and its receptor (GnRHR) are both expressed by a number of malignant tumors, including those of the breast. In the latter, both behave as potent inhibitors of invasion. Nevertheless, the signaling pathways whereby the activated GnRH/GnRHR system exerts this effect have not been clearly established. In this study, we provide experimental evidence that describes components of the mechanism(s) whereby GnRH inhibits breast cancer cell invasion. METHODS: Actin polymerization and substrate adhesion was measured in the highly invasive cell line, MDA-MB-231 transiently expressing the wild-type or mutant DesK191 GnRHR by fluorometry, flow cytometric analysis, and confocal microscopy, in the absence or presence of GnRH agonist. The effect of RhoA-GTP on stress fiber formation and focal adhesion assembly was measured in MDA-MB-231 cells co-expressing the GnRHRs and the GAP domain of human p190Rho GAP-A or the dominant negative mutant GAP-Y1284D. Cell invasion was determined by the transwell migration assay. RESULTS: Agonist-stimulated activation of the wild-type GnRHR and the highly plasma membrane expressed mutant GnRHR-DesK191 transiently transfected to MDA-MB-231 cells, favored F-actin polymerization and substrate adhesion. Confocal imaging allowed detection of an association between F-actin levels and the increase in stress fibers promoted by exposure to GnRH. Pull-down assays showed that the effects observed on actin cytoskeleton resulted from GnRH-stimulated activation of RhoA GTPase. Activation of this small G protein favored the marked increase in both cell adhesion to Collagen-I and number of focal adhesion complexes leading to inhibition of the invasion capacity of MDA-MB-231 cells as disclosed by assays in Transwell Chambers. CONCLUSIONS: We here show that GnRH inhibits invasion of highly invasive breast cancer-derived MDA-MB-231 cells. This effect is mediated through an increase in substrate adhesion promoted by activation of RhoA GTPase and formation of stress fibers and focal adhesions. These observations offer new insights into the molecular mechanisms whereby activation of overexpressed GnRHRs affects cell invasion potential of this malignant cell line, and provide opportunities for designing mechanism-based adjuvant therapies for breast cancer.

Novel mutation in the gonadotropin-releasing hormone receptor (GNRHR) gene in a patient with normosmic isolated hypogonadotropic hypogonadism / Nova mutação no receptor do hormônio liberador de gonadotrofinas (GNRHR) em um paciente com hipogonadismo hipogonadotrófico isolado normósmico

We report a novel GNRHR mutation in a male with normosmic isolated hypogonadotropic hypogonadism (nIHH). The coding region of the GNRHR gene was amplified and sequenced. Three variants p.[Asn10Lys;Gln11Lys]; [Tyr283His] were identified in the GNRHR coding region in a male with sporadic complete nIHH. The three variants were absent in the controls (130 normal adults). Familial segregation showed that the previously described p.Asn10Lys and p.Gln11Lys are in the same allele, in compound heterozygozity with the novel variant p.Tyr283His. The p.[Asn10Lys;Gln11Lys] are known inactivating mutations. The p.Tyr283His affects a well-conserved residue, and in silico analysis suggested it is a deleterious variant. We describe a novel GNRHR mutation in a male with nIHH. Absence of the mutation in the control group, conservation among species, in silico analysis, and familial segregation suggest that p.Tyr283His, which was identified in compound heterozygozity with the p.[Asn10Lys;Gln11Lys] variants, is an inactivating mutation. Arq Bras Endocrinol Metab. 2012;56(8):540-4.

Relatamos uma nova mutação no gene GNRHR em um homem com hipogonadismo hipogonadotrófico isolado normósmico (HHIn). A região codificadora do gene GNRHR foi amplificada e sequenciada. Três variantes p.[Asn10Lys;Gln11Lys]; [Tyr283His] foram identificadas no GNRHR em um homem com HHIn esporádico. As três variantes estavam ausentes no grupo controle (130 adultos normais). A segregação familiar mostrou que as variantes previamente descritas p.[Asn10Lys;Gln11Lys] se localizavam no mesmo alelo, em heterozigose composta com a nova variante p.Tyr283His. As mutações p.[Asn10Lys;Gln11Lys] são sabidamente inativadoras. A variante p.Tyr283His afeta um resíduo bem conservado, e a análise in silico sugeriu que essa é uma mutação deletéria. Descrevemos uma mutação inédita no gene GNRHR em um paciente com HHIn nIHH. A ausência da variante no grupo controle, a conservação entre as espécies, a análise in silico e a segregação familiar sugerem que a p.Tyr283His é uma mutação inativadora, identificada em heterozigose composta com as mutações p.[Asn10Lys;Gln11Lys]. Arq Bras Endocrinol Metab. 2012;56(8):540-4.

Association of neuropeptide Y and gonadotrophin-releasing hormone receptor gene SNPs with breeding value for growth and egg production traits in Mazandaran native chickens.

Neuropeptide Y (NPY) and gonadotrophin-releasing hormone receptor (GnRHR) are two candidate genes with a wide variety of physiological functions in growth and especially in reproduction processes. We examined the association of one SNP from each of these genes with growth- and egg production-related traits in Mazandaran native chickens. Two hundred and six individuals were genotyped by PCR-RFLP. Marker-trait association analyses were performed using both breeding value and phenotypic information. The data came from 18 successive generations of selection at a Mazandaran native chicken breeding station in Iran. Data were analyzed with a univariate animal model in an ASREML procedure to estimate breeding values of the birds for these traits. Two alleles were found for both genes, A and a alleles for GnRHR, with frequencies of 0.614 and 0.386, B and b alleles for NPY, with frequencies of 0.780 and 0.221, respectively. The additive genetic effects of the GnRHR gene on egg number and egg mass were significant. Also, body weight at sexual maturity was significantly influenced by the NPY gene. We conclude that GnRHR and NPY genes are associated with egg production and growth traits, respectively.

Association between GNRHR, LHR and IGF1 polymorphisms and timing of puberty in male Angus cattle.

BACKGROUND: In bovines, there are significant differences within and among beef breeds in the time when bulls reach puberty. Although the timing of puberty is likely to be a multigenic trait, previous studies indicate that there may also be single genes that exert major effects on the timing of puberty within the general population. Despite its economic importance, there are not many SNPs or genetic markers associated with the age of puberty in male cattle. In the present work, we selected three candidate genes, GNRHR, LHR and IGF1, and associated their polymorphisms with the age of puberty in Angus male cattle. RESULTS: After weaning, 276 Angus males were measured every month for weight (W), scrotal circumference (SC), sperm concentration (C) and percentage of motility (M). A total of 4 SNPs, two within GNRHR, one in LHR and one in IGF1 were genotyped using the pyrosequencing technique. IGF1-SnaBI SNP was significant associated (P < 0.01) with age at SC 28 cm, but it were not associated with age at M 10% and C 50 million. Genotype CC exhibited an average age at SC 28 cm of 7 and 11 days higher than CT (p = 0.037) and TT (p = 0.012), respectively. This SNP explained 1.5% of the genetic variance of age of puberty at SC28. LHR-I499L, GNRHR-SNP5 and GNRHR-SNP6 were not associated with any of the measurements. However, GNRHR haplotypes showed a suggestive association with age at SC 28 cm. CONCLUSIONS: The findings presented here could support the hypothesis that IGF1 is a regulator of the arrival to puberty in male calves and is involved in the events that precede and initiate puberty in bull calves. Given that most studies in cattle, as well as in other mammals, were done in female, the present results are the first evidence of markers associated with age at puberty in male cattle.

Novel mutation in the gonadotropin-releasing hormone receptor (GNRHR) gene in a patient with normosmic isolated hypogonadotropic hypogonadism.

We report a novel GNRHR mutation in a male with normosmic isolated hypogonadotropic hypogonadism (nIHH). The coding region of the GNRHR gene was amplified and sequenced. Three variants p.[Asn10Lys;Gln11Lys]; [Tyr283His] were identified in the GNRHR coding region in a male with sporadic complete nIHH. The three variants were absent in the controls (130 normal adults). Familial segregation showed that the previously described p.Asn10Lys and p.Gln11Lys are in the same allele, in compound heterozygozity with the novel variant p.Tyr283His. The p.[Asn10Lys;Gln11Lys] are known inactivating mutations. The p.Tyr283His affects a well-conserved residue, and in silico analysis suggested it is a deleterious variant. We describe a novel GNRHR mutation in a male with nIHH. Absence of the mutation in the control group, conservation among species, in silico analysis, and familial segregation suggest that p.Tyr283His, which was identified in compound heterozygozity with the p.[Asn10Lys;Gln11Lys] variants, is an inactivating mutation.

Pharmacoperones: a new therapeutic approach for diseases caused by misfolded G protein-coupled receptors.

G Protein-coupled receptors (GPCRs) are cell membrane proteins that recognize specific chemical signals such as drugs and hormones and transduce these signals into cellular responses by activating G-proteins. As is the case for all newly synthesized proteins, GPCRs are subjected to conformational scrutiny at the endoplasmic reticulum prior to processing and trafficking to the cell surface membrane. Because of this stringent quality control screening mechanism, mutations that result in protein misfolding frequently lead to retention in the endoplasmic reticulum, aggregation or other misrouting and, eventually, to disease. This article reviews some patents and new therapeutic opportunities based on the misfolding and retention of otherwise functional GPCRs that represent promising approaches to correct conformational abnormalities leading to distinct disease states.

Polymorphisms associated with egg number at 300 days of age in chickens.

We looked for variations that could be associated with chicken egg number at 300 days of age (EN300) in seven genes of the hypothalamic-pituitary-gonadal axis, including gonadotrophin-releasing hormone-I (GnRH-I), GnRH receptor (GnRHR), neuropeptide Y (NPY), dopamine D2 receptor (DRD2), vasoactive intestinal polypeptide (VIP), VIP receptor-1 (VIPR-1), prolactin (PRL), and the QTL region between 87 and 105 cM of the Z chromosome. Ten mutations in the seven genes were chosen to do marker-trait association analyses in a population comprising 1310 chickens, which were obtained from a company located in Guangdong Province of China. The C1704887T of VIPR-1 was found to have a highly significant association with EN300. The T5841629C of DRD2 and the C1715301T of VIPR-1 were significantly associated with EN300. A highly significant association was also found between the C1704887T-C1715301T haplotypes of VIPR-1 and EN300. H1H3 had the highest EN300. Four PCR-RFLP variations in the candidate QTL region were selected to investigate their genetic effects on EN300. The haplotypes of T32742468C-G32742603A in this region showed a highly significant association with EN300. Bioinformatics analyses showed that both T32742468C and G32742603A were located in intron 1 of the SH3-domain GRB2-like 2 (SH3GL2) gene. We conclude that five SNPs, including C1704887T and C1715301T of VIPR-1, T5841629C of DRD2, and T32742468C and G32742603A of SH3GL2, would be useful as markers for breeding to increase chicken EN300.

The neuropeptide Gonadotropin-releasing hormone modifies the spontaneous muscular contraction in the earthworm: Eisenia foetida.

We investigated whether the Gonadotropin-releasing hormone affects the spontaneous muscular contraction in the earthworm Eisenia foetida. In addition, we investigated the presence of Gonadotropin-releasing hormone receptor in ventral nerve cord by immunohistochemistry and polymerase chain reaction. Gonadotropin-releasing hormone induced a significant increase on both amplitude and muscular tone and decrease in the frequency of spontaneous muscular contraction. We found the presence of immunoreactive material to Gonadotropin-releasing hormone receptor in the ventral nerve cord, likewise the Gonadotropin-releasing hormone receptor mRNA expression. In conclusion, the Gonadotropin-releasing hormone modifies the spontaneous muscular contraction in E. foetida and these effects can be due to the activation of the Gonadotropin-releasing hormone receptor.

Biochemical mechanism of pathogenesis of human gonadotropin-releasing hormone receptor mutants Thr104Ile and Tyr108Cys associated with familial hypogonadotropic hypogonadism.

The pathogenic mechanisms whereby the Thr104Ile and Tyr108Cys mutations in the gonadotropin-releasing hormone receptor (GnRHR) gene cause hypogonadotropic hypogonadism in humans are unknown. Transient expression of Thr104Ile and Tyr108Cys mutants in COS-7 cells revealed that both GnRHR mutants neither bind nor respond to agonist. Removal of Lys191 rescued function of both mutants, while addition of a carboxyl-terminal targeting sequence only rescued function of the Thr104Ile mutant. Exposure to the pharmacoperone In3 rescued almost completely Thr104Ile mutant function to wild-type levels, whereas rescue was partial for the Tyr108Cys GnRHR. Additional mutations that block formation of bridges involving Cys108 showed that a Cys108-Cys200 disulfide bridge is the predominant moiety formed in the Tyr108Cys mutant. Thr104Ile and Tyr108Cys GnRHRs are misfolded structures whose function is rescuable by genetic and/or pharmacological strategies. The Tyr108Cys mutant forms an aberrant disulfide bridge that prevents formation of the required Cys14-Cys200 bridge essential for GnRHR plasma membrane expression.