An N-ethyl-n-nitrosourea induced corticotropin-releasing hormone promoter mutation provides a mouse model for endogenous glucocorticoid excess

Cushing's syndrome, which is characterized by excessive circulating glucocorticoid concentrations, maybe due to ACTH-dependent or -independent causes that include anterior pituitary and adrenal cortical tumors, respectively. ACTH secretion is stimulated by CRH, and we report a mouse model for Cushing's syndrome due to an N-ethyl-N-nitrosourea (ENU) induced Crh mutation at -120 bp of the promoter region, which significantly increased luciferase reporter activity and was thus a gain-of-function mutation. Crh -120/+ mice, when compared with wild-type littermates, had obesity, muscle wasting, thin skin, hair loss, and elevated plasma and urinary concentrations of corticosterone. In addition, Crh-120/+ mice had hyperglycemia, hyperfructosaminemia, hyperinsulinemia, hypercholesterolemia, hypertriglyceridemia, and hyperleptinemia but normal adiponectin. Crh -120/+ mice also had low bone mineral density, hypercalcemia, hypercalciuria, and decreased concentrations of plasma PTH and osteocalcin. Bone histomorphometry revealed Crh-120/+ mice to have significant reductions in mineralizing surface area, mineral apposition, bone formation rates, osteoblast number, and the percentage of corticoendosteal bone covered by osteoblasts, which was accompanied by an increase in adipocytes in the bone marrow. Thus, a mouse model for Cushing's syndrome has been established, and this will help in further elucidating the pathophysiological effects of glucocorticoid excess and in evaluating treatments for corticosteroid-induced osteoporosis.

Regulation of Gonadotropin-Releasing Hormone (GnRH)-Receptor Gene Expression in Tilapia: Effect of GnRH and Dopamine

The present work was designed to study certain aspects of the endocrine regulation of gonadotropin-releasing hormone receptor (GnRH-R) in the pituitary of the teleost fish tilapia. A GnRH-R was cloned from the pituitary of hybrid tilapia (taGnRH-R) and was identified as a typical seven-transmembrane receptor. Northern blot analysis revealed a single GnRH-R transcript in the pituitary of approximately 2.3 kilobases. The taGnRH-R mRNA levels were significantly higher in females than in males. Injection of the salmon GnRH analog (sGnRHa; 5–50 μg/kg) increased the steady-state levels of taGnRH-R mRNA, with the highest response recorded at 25 μg/kg and at 36 h. At the higher dose of sGnRHa (50 μg/kg), taGnRH-R transcript appeared to be down-regulated. Exposure of tilapia pituitary cells in culture to graded doses (0.1–100 nM) of seabream (sbGnRH = GnRH I), chicken II (cGnRH II), or salmon GnRH (sGnRH = GnRH III) resulted in a significant increase in taGnRH-R mRNA levels. The highest levels of both LH release and taGnRH-R mRNA levels were recorded after exposure to cGnRH II and the lowest after exposure to sbGnRH. The dopamine-agonist quinpirole suppressed LH release and mRNA levels of taGnRH-R, indicating an inhibitory effect on GnRH-R synthesis. Collectively, these data provide evidence that GnRH in tilapia can up- regulate, whereas dopamine down-regulates, taGnRH-R mRNA levels.

A role for calcium in gonadotrophin-releasing hormone (GnRH) stimulated secretion of chorionic gonadotrophin by first trimester human placental minces in vitro

In vitro studies using first-trimester human placental minces have shown that stimulation of human chorionic gonadotrophin (hCG) secretion by gonadotrophin-releasing hormone (GnRH) is dependent upon the presence of extracellular calcium. Addition of GnRH to first-trimester placental minces in vitro was found to stimulate 45Ca2+ uptake into placental minces, and the process was associated with an increase in immunoreactive hCG in the medium. Addition of GnRH to placental minces preloaded with 45Ca2+ stimulated the efflux of 45Ca2+ within one minute. The calmodulin inhibitors chlorpromazine andtrifluoperazine inhibited the basal uptake and efflux of 45Ca2+ suggesting the involvement of calmodulin in the mobilization of calcium in the placenta.

Use of adult male bonnet monkeys (Macaca radiata) for testing the biological activity of gonadotropin releasing hormone and its analogues

The effect of injecting agonistic and antagonistic analogues of gonadotropin releasing hormone analogues on serum testosterone levels was checked in adult and immature male bonnet monkeys. Of the agonistic analogues Buserelin, Ovurelin and D-Phe6 Gln8 GnRH were found to be most potent in increasing serum testosterone levels in the adult male bonnet monkeys. While 27-month-old monkeys responded well to des Gly10 GnRH, only marginal response was observed in the case of 15-month-old monkeys. Studies carried out with Ovurelin indicated that it was not effective in causing desensitization in adult monkeys. The antagonistic analogue was effective in blocking nocturnal surge of serum testosterone. Based on these studies it is suggested the adult male bonnet monkeys can be effectively used for testing the activity of GnRH analogues.

Chronic suppression of testicular function by constant infusion of gonadotropin-releasing hormone agonist and testosterone supplementation in the bonnet monkey (Macaca radiata)

Objective: To study the efficacy of long-term buserelin acetate infusion to desensitize pituitary and block testicular function in adult male monkeys (Macaca radiata). Animals: Proven fertile male monkeys exhibiting normal testicular function. Protocol: Each of the control (n = 5) and experimental monkeys (n = 10) received a fresh miniosmotic pump every 21 days, whereas pumps in controls delivered vehicle of experimentals released 50-mu-g buserelin acetate every 24 hours. On day 170 (renewed every 60 days) a silastic capsule containing crystalline testosterone (T) was implanted in the experimental monkeys. At the end of 3 years, treatment was stopped, and recovery of testicular function and fertility monitored. Results: (1) Treatment resulted in marked reduction of nocturnal but not basal serum T; (2) the pituitary remained desensitized to buserelin acetate throughout the 3-year period; (3) animals were largely azoospermic with occasional oligospermia exhibited by two monkeys; and (4) withdrawal of treatment restored testicular function, with 70% of animals regaining fertility. Conclusion: Long-term infertility (but restorable) can be induced in male monkeys by constant infusion of buserelin acetate and T.

Effect of constant infusion of gonadotropin releasing hormone (GnRH) agonist Buserelin and antagonist CDB 2085 A using osmotic minipumps on testicular function in adult male bonnet monkey (Macaca radiata)

The effect of chronic infusion of gonadotropic hormone agonist Buserelin or antagonist CDB 2085 A for 15 weeks via alzet minipumps in adult male bonnet monkeys was studied. Infusion of Buserelin resulted in a decrease in the difference between serum testosterone values at 22.00 hours and 10.00 hours, decrease in responsiveness to injected Buserelin as judged by change in serum testosterone values from pre-injection values and decrease in sperm counts. Infusion of antagonist resulted in a decrease in the difference between serum testosterone values at 22.00 hours and 10.00 hours.

The secretion of gonadotrophin-releasing hormone by peri-implantation embryos of the rhesus monkey: comparison with the secretion of chorionic gonadotrophin

Chorionic gonadotrophin (CG) is the first clear embryonic signal during early pregnancy in primates. CG has close structural and functional similarities to pituitary luteinizing hormone (LH) which is regulated by gonadotrophin releasing hormone (GnRH). To study the regulatory mechanism of CG secretion in primate embryos, we examined the production and timing of secretion of GnRH in peri-implantation embryos of the rhesus monkey. In-vivo fertilized/developed morulae and early blastocysts, recovered from non-superovulated, naturally-bred rhesus monkeys by non-surgical uterine flushing, were cultured in vitro to hatched, attached and post-attached blastocyst stages using a well-established culture system. We measured GnRH and CG in media samples from cultured embryos with a sensitive radioimmunoassay and bioassay, respectively. The secretion of GnRH (pg/ml; mean +/- SEM) by embryos (n = 20) commenced from low levels (0.32 +/- 0.05) during the pre-hatching blastocyst stage to 0.70 +/- 0.08 at 6-12 days and 1.30 +/- 0.23 at greater than or equal to 13 days of hatched blastocyst attachment and proliferation of trophoblast cells. GnRH concentrations in culture media obtained from embryos (n = 5) that failed to hatch and attach were mostly undetectable (less than or equal to 0.1). Samples that did not contain detectable GnRH failed to show detectable CG. Immunocytochemical studies, using a specific monoclonal anti-GnRH antibody (HU4H) as well as polyclonal antisera (LR-1), revealed that immunopositive GnRH cells were localized in pre-hatching blastocysts (n = 4), in blastocysts (n = 2) after 5-10 days of attachment and in monolayer cultures (n = 4) of well-established embryonic trophoblast cells. GnRH positive staining was seen only in cytotrophoblasts but not in syncytiotrophoblasts. Similarly, cytotrophoblast, but not syncytiotrophoblast, cells of the rhesus placenta were immunopositive. In controls, either in the absence of antibody or in the presence of antibody pre-absorbed with GnRH, these cells failed to show stain. These observations indicate, for the first time, that an immunoreactive GnRH is produced and secreted by blastocysts during the peri-attachment period and by embryo-derived cytotrophoblast cells in the rhesus monkey.

Molecular mimicry by antiidiotypic monoclonal antibody to gonadotropin releasing hormone

Antipeptide and antiidiotypic antibodies to several receptors are known to mimic their respective ligands in transducing signals on binding their receptors. In our attempts to study gonadotropin releasing hormone receptor, antipeptide and antiidiotypic monoclonal antibodies specific to the receptor were established earlier. The antipeptide mAb F1G4 was to a synthetic peptide corresponding to the extracellular domain of human GnRH receptor and the antiidiotypic mAb 4D10C1 was to the idiotype of a GnRH specific mAb. Here we report the physiological effects of the two mAbs on binding the receptor, as investigated using in vitro cultures of(a) human term placental villi and (b) rat pituitaries. The mAb 4D10C1 exerted a dose-dependent release of human chorionic gonadonopin in cultures of human term placental villi as well as luteinising and follicle stimulating hormones in cultures of rat pituitaries.

l-pGlu-(2-propyl)-l-His-l-ProNH2 attenuates 4-aminopyridine-induced epileptiform activity and sodium current: a possible action of new thyrotropin-releasing hormone analog for its anticonvulsant potential

L-PGlu-(2-proPyl)-L-His-L-ProNH(2) (NP-647) is a CNS active thyrotropin-releasing hormone (TRH) analog with potential application in various CNS disorders including seizures. In the present study, mechanism of action for protective effect of NP-647 was explored by studying role of NP-647 on epileptiform activity and sodium channels by using patch-clamp methods. Epileptiform activity was induced in subicular pyramidal neurons of hippocampal slice of rat by perfusing 4-aminopyridine (4-AP) containing Mg(+2)-free normal artificial cerebrospinal fluid (nACSF). Increase in mean firing frequency was observed after perfusion of 4-AP and zero Mg(+2) (2.10+/-0.47 Hz) as compared with nACSF (0.12+/-0.08 Hz). A significant decrease in mean firing frequency (0.61+/-0.22 Hz), mean frequency of epileptiform events (0.03+/-0.02 Hz vs. 0.22+/-0.05 Hz of 4-AP+0 Mg), and average number of action potentials in paroxysmal depolarization shift-burst (2.54+/-1.21 Hz vs. 8.16+/-0.88 Hz of 4-AP +0 Mg) was observed. A significant reduction in peak dV/dt (246+/-19 mV ms(-1) vs. 297 18 mV ms-1 of 4-AP+0 Mg) and increase (1.332+/-0.018 ms vs. 1.292+/-0.019 ms of 4-AP+0 Mg) in time required to reach maximum depolarization were observed indicating role of sodium channels. Concentration-dependent depression of sodium current was observed after exposure to dorsal root ganglion neurons to NP-647. NP-647 at different concentrations (1, 3, and 10 mu M) depressed sodium current (15+/-0.5%, 50+/-2.6%, and 75+/-0.7%, respectively). However, NP-647 did not show change in the peak sodium current in CNa18 cells. Results of present study demonstrated potential of NP-647 in the inhibition of epileptiform activity by inhibiting sodium channels indirectly. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Antisense for gonadotropin-releasing hormone reduces gonadotropin synthesis and gonadal development in transgenic common carp (Cyprinus carpio)

Generating transgenic fish with desirable traits (e.g., rapid growth, larger size, etc.) for commercial use has been hampered by concerns for biosafety and competition if these fish are released into the environment. These obstacles may be overcome by producing transgenic fish that are sterile, possibly by inhibiting hormones related to reproduction. In vertebrates, synthesis and release of gonadotropin (GtH) and other reproductive hormones is mediated by gonadotropin-releasing hormone (GnRH). Recently two cDNA sequences encoding salmon-type GnRH (sGnRH) decapeptides were cloned from common carp (Cyprinus carpio). This study analyzed the expression of these two genes using real-time polymerase chain reaction (RT-PCR) in different tissues carp at varying developmental stages. Transcripts of both genes were detected in ovary and testis in mature and regressed, but not in juvenile carp. To evaluate the effects of sGnRH inhibition, the recombinant gene CAsGnRHpc-antisense, expressing antisense sGnRH RNA driven by a carp beta-actin promoter, was constructed. Blocking sGnRH expression using antisense sGnRH significantly decreased GtH in the blood of male transgenic carp. Furthermore, some antisense transgenic fish had no gonadal development and were completely sterile. These data demonstrate that sGnRH is important for GtH synthesis and development of reproductive organs in carp. Also, the antisense sGnRH strategy may prove effective in generating sterile transgenic fish, eliminating environmental concerns these fish may raise. (c) 2007 Published by Elsevier B.V.

Alpha-fetoprotein controls female fertility and prenatal development of the gonadotropin-releasing hormone pathway through an antiestrogenic action

It has been shown previously that female mice homozygous for an alpha-fetoprotein (AFP) null allele are sterile as a result of anovulation, probably due to a defect in the hypothalamic-pituitary axis. Here we show that these female mice exhibit specific anomalies in the expression of numerous genes in the pituitary, including genes involved in the gonadotropin-releasing hormone pathway, which are underexpressed. In the hypothalamus, the gonadotropin-releasing hormone gene, Gnrh1, was also found to be down-regulated. However, pituitary gene expression could be normalized and fertility could be rescued by blocking prenatal estrogen synthesis using an aromatase inhibitor. These results show that AFP protects the developing female brain from the adverse effects of prenatal estrogen exposure and clarify a long-running debate on the role of this fetal protein in brain sexual differentiation.