Physiological and pharmacological overview of the gonadotropin releasing hormone.

Gonadotropin-Releasing Hormone (GnRH) is a decapeptide responsible for the control of the reproductive functions. It shows C- and N-terminal aminoacid modifications and two other distinct isoforms have been so far identified. The biological effects of GnRH are mediated by binding to high-affinity G-protein couple receptors (GnRHR), showing characteristic very short C tail. In mammals, including humans, GnRH-producing neurons originate in the embryonic nasal compartment and during early embryogenesis they undergo rapid migration towards the hypothalamus; the increasing knowledge of such mechanisms improved diagnostic and therapeutic approaches to infertility. The pharmacological use of GnRH, or its synthetic peptide and non-peptide agonists or antagonists, provides a valid tool for reproductive disorders and assisted reproduction technology (ART). The presence of GnRHR in several organs and tissues indicates additional functions of the peptide. The identification of a GnRH/GnRHR system in the human endometrium, ovary, and prostate has extended the functions of the peptide to the physiology and tumor transformation of such tissues. Likely, the activity of a GnRH/GnRHR system at the level of the hippocampus, as well as its decreased expression in mice brain aging, raised interest in its possible involvement in neurogenesis and neuronal functions. In conclusion, GnRH/GnRHR appears to be a fascinating biological system that exerts several possibly integrated pleiotropic actions in the complex control of reproductive functions, tumor growth, neurogenesis, and neuroprotection. This review aims to provide an overview of the physiology of GnRH and the pharmacological applications of its synthetic analogs in the management of reproductive and non-reproductive diseases.

Complementary effectiveness of carotid sinus massage and tilt testing for the diagnosis of reflex syncope in patients older than 40 years: a cohort study.

AIMS: Indications, methodology, and diagnostic criteria for carotid sinus massage (CSM) and tilt testing (TT) have been standardized by the 2018 Guidelines on Syncope of the European Society of Cardiology. Aim of this study was to assess their effectiveness in a large cohort which reflects the performance under 'real-world' conditions. METHODS AND RESULTS: We analysed all patients who had undergone CSM and TT in the years 2003-2019 for suspected reflex syncope. Carotid sinus massage was performed according to the 'Method of Symptoms'. Tilt testing was performed according to the 'Italian protocol' which consists of a passive phase followed by a sublingual nitroglycerine phase. For both tests, positive test was defined as reproduction of spontaneous symptoms in the presence of bradycardia and/or hypotension. Among 3293 patients (mean age 73 ± 12 years, 48% males), 2019 (61%) had at least one test positive. A bradycardic phenotype was found in 420 patients (13%); of these, 60% were identified by CSM, 37% by TT, and 3% had both test positive. A hypotensive phenotype was found in 1733 patients (53%); of these, 98% were identified by TT and 2% had both TT and CSM positive. CONCLUSION: The overall diagnostic yield of the tests in patients >40-year-old with suspected reflex syncope was 61%. Both CSM and TT are useful for identifying those patients with a bradycardic phenotype, whereas CSM has a limited value for identifying the hypotensive phenotype. Since the overlap of responses between tests is minimal, both CSM and TT should be performed in every patient over 40 years receiving investigation for unexplained but possible reflex syncope.

Food for Bone: Evidence for a Role for Delta-Tocotrienol in the Physiological Control of Osteoblast Migration.

Bone remodeling and repair require osteogenic cells to reach the sites that need to be rebuilt, indicating that stimulation of osteoblast migration could be a promising osteoanabolic strategy. We showed that purified δ-tocotrienol (δ-TT, 10 µg/mL), isolated from commercial palm oil (Elaeis guineensis) fraction, stimulates the migration of both MC3T3-E1 osteoblast-like cells and primary human bone marrow mesenchymal stem cells (BMSC) as detected by wound healing assay or Boyden chamber assay respectively. The ability of δ-TT to promote MC3T3-E1 cells migration is dependent on Akt phosphorylation detected by Western blotting and involves Wnt/ß-catenin signalling pathway activation. In fact, δ-TT increased ß-catenin transcriptional activity, measured using a Nano luciferase assay and pretreatment with procaine (2 µM), an inhibitor of the Wnt/ß-catenin signalling pathway, reducing the wound healing activity of δ-TT on MC3T3-E1 cells. Moreover, δ-TT treatment increased the expression of ß-catenin specific target genes, such as Osteocalcin and Bone Morphogenetic Protein-2, involved in osteoblast differentiation and migration, and increased alkaline phosphatase and collagen content, osteoblast differentiation markers. The ability of δ-TT to enhance the recruitment of BMSC, and to promote MC3T3-E1 differentiation and migratory behavior, indicates that δ-TT could be considered a promising natural anabolic compound.

GnRH and GnRH receptors in the pathophysiology of the human female reproductive system.

BACKGROUND: Human reproduction depends on an intact hypothalamic-pituitary-gonadal (HPG) axis. Hypothalamic gonadotrophin-releasing hormone (GnRH) has been recognized, since its identification in 1971, as the central regulator of the production and release of the pituitary gonadotrophins that, in turn, regulate the gonadal functions and the production of sex steroids. The characteristic peculiar development, distribution and episodic activity of GnRH-producing neurons have solicited an interdisciplinary interest on the etiopathogenesis of several reproductive diseases. The more recent identification of a GnRH/GnRH receptor (GnRHR) system in both the human endometrium and ovary has widened the spectrum of action of the peptide and of its analogues beyond its hypothalamic function. METHODS: An analysis of research and review articles published in international journals until June 2015 has been carried out to comprehensively summarize both the well established and the most recent knowledge on the physiopathology of the GnRH system in the central and peripheral control of female reproductive functions and diseases. RESULTS: This review focuses on the role of GnRH neurons in the control of the reproductive axis. New knowledge is accumulating on the genetic programme that drives GnRH neuron development to ameliorate the diagnosis and treatment of GnRH deficiency and consequent delayed or absent puberty. Moreover, a better understanding of the mechanisms controlling the episodic release of GnRH during the onset of puberty and the ovulatory cycle has enabled the pharmacological use of GnRH itself or its synthetic analogues (agonists and antagonists) to either stimulate or to block the gonadotrophin secretion and modulate the functions of the reproductive axis in several reproductive diseases and in assisted reproduction technology. Several inputs from other neuronal populations, as well as metabolic, somatic and age-related signals, may greatly affect the functions of the GnRH pulse generator during the female lifespan; their modulation may offer new possible strategies for diagnostic and therapeutic interventions. A GnRH/GnRHR system is also expressed in female reproductive tissues (e.g. endometrium and ovary), both in normal and pathological conditions. The expression of this system in the human endometrium and ovary supports its physiological regulatory role in the processes of trophoblast invasion of the maternal endometrium and embryo implantation as well as of follicular development and corpus luteum functions. The GnRH/GnRHR system that is expressed in diseased tissues of the female reproductive tract (both benign and malignant) is at present considered an effective molecular target for the development of novel therapeutic approaches for these pathologies. GnRH agonists are also considered as a promising therapeutic approach to counteract ovarian failure in young female patients undergoing chemotherapy. CONCLUSIONS: Increasing knowledge about the regulation of GnRH pulsatile release, as well as the therapeutic use of its analogues, offers interesting new perspectives in the diagnosis, treatment and outcome of female reproductive disorders, including tumoral and iatrogenic diseases.

Assessment of the vasodepressor reflex in carotid sinus syndrome.

BACKGROUND: Assessment of the vasodepressor reflex in carotid sinus syndrome is influenced by the method of execution of the carotid sinus massage and the coexistence of the cardioinhibitory reflex. METHODS AND RESULTS: Carotid sinus massage reproduced spontaneous symptoms in 164 patients in the presence of hypotension or bradycardia (method of symptoms). When an asystolic pause was induced, the vasodepressor reflex was reassessed after suppression of the asystolic reflex by means of 0.02 mg/kg IV atropine. An isolated vasodepressor form was found in 32 (20%) patients, who had lowest systolic blood pressure (SBP) of 65±15 mm Hg. Of these, only 21 (66%) patients had an SBP fall ≥50 mm Hg, which is the universally accepted cut-off value for the diagnosis of the vasodepressor form. Conversely, a lowest SBP value of ≤85 mm Hg (corresponding to the fifth percentile) detected 97% of vasodepressor patients, but was also present in 84% of the 132 patients with an asystolic reflex. These latter patients had both asystole ≥3 s (mean 7.6±2.2 s) and SBP fall to 63±22 mm Hg: in 46 (28%) patients, symptoms persisted after atropine (mixed form), in the remaining 86 (52%) patients, symptoms did not (cardioinhibitory form) persist. CONCLUSIONS: The current definition of ≥50 mm Hg SBP fall failed to identify one third of patients with isolated vasodepressor form. A cut-off value of symptomatic SBP of ≤85 mm Hg seems more appropriate, but it is unable to identify cardioinhibitory forms. In asystolic forms, atropine testing is able to distinguish a cardioinhibitory form from a mixed form.

Resynchronization of the left ventricular contraction by tailored programming of right and left ventricular pacing.

AIMS: The prerequisite and the rationale for the benefit of cardiac resynchronization therapy (CRT) is that it is able to resynchronize left ventricular (LV) walls that have a delayed activation. METHODS AND RESULTS: In 69 consecutive patients who underwent biventricular (BIV) pacemaker implantation, we assessed the magnitude of intraventricular resynchronization achieved by means of simultaneous (BIV 0) and sequential BIV pacing (with an individually optimized VV interval value among +80 ms and -80 ms) using pulsed-wave tissue Doppler imaging techniques and in particular the measurement of the intra-LV electromechanical delay. The intra-LV delay was defined as the difference between the longest and the shortest activation time in the six basal segments of the LV. An abnormal intra-LV delay was defined as a value >41 ms. The intra-LV delay was 63 +/- 28 ms baseline, decreased to 44 +/- 26 ms with BIV 0 and to 26 +/- 15 ms with optimized BIV (P = 0.001). BIV 0 determined the shortest delay in 28 (41%) patients (23 +/- 12 ms). In 41 (59%) patients, a better resynchronization was achieved with optimized VV intervals (LV first in 32 and RV first in 5) or single-chamber pacing (LV in 3 and RV in 1). With BIV 0, the intra-LV delay remained abnormal in 41% and was longer than baseline in 30% of patients compared with 9 and 12% with optimized BIV, respectively (P = 0.001). CONCLUSION: A sub-optimal resynchronization is achieved with simultaneous BIV pacing in most patients. A tailored programming of the relative contribution of RV and LV pacing forms the prerequisite for improving CRT results.

Presence of delta opioid receptors on a subset of hypothalamic gonadotropin releasing hormone (GnRH) neurons.

Opioid peptides exert an inhibitory effect on hypothalamic gonadotropin releasing hormone (GnRH) secretion mainly by interacting with mu-opioid receptors. Although a direct role for opioids via delta-opioid receptors (DORs) has been suggested, the presence of these receptors on GnRH neurons has never been demonstrated. In the present study, we determined the distribution of DORs in the basal hypothalamus of rat with special focus on their relation to GnRH neurons. Double-labelling immunofluorescence and confocal microscopy revealed that DORs are exclusively present in a subpopulation of GnRH nerve terminals, with the highest density in the external layer of the median eminence. We then studied the functional characteristics of DORs in an immortalized GnRH-secreting neuronal cell line (GT1-1) known to endogenously express this receptor. Here, pertussis toxin pretreatment abolished the delta-agonist (DPDPE) inhibitory effect on cAMP accumulation. We also analyzed the type of G proteins involved in the signal transduced by the DOR and showed that GT1-1 cells express the inhibitory Go and Gi2 alpha-subunits. However, only Go was down-regulated under chronic DPDPE exposure. Finally, since DOR is expressed postnatally in brain, we compared GnRH neuronal cells immortalized at different developmental stages (the more mature GT1-1 and GT1-7 cells, versus the more immature GN11 cells), evidencing that only mature neurons express DOR. In conclusion, our study indicates that a direct control of opioids via delta-receptors occurs on GnRH neurons and validates the use of GT1 cells to further investigate the nature of the DOR present on GnRH neurons.

Biologia de neurônios hipotalâmicos produtores do hormônio liberador das gonadotrofinas: novas informações sobre o uso das células imortalizadas secretoras desse hormônio / Biology of hypothalamic neurons producing gonadotropin releasing hormone (GnRH): new information on the use of immortalized cells secreting GnRH

O estudo dos neurônios que produzem o hormônio liberador das gonadotrofinas (GnRH), hormônio hipotalámico que estimula a secreção, das gonadotrofinas hipofisárias, tem recebido vigoroso impulso com a disponibilidade das células imortalizadas, que especificamente sintetizam e secretam o hormônio em questão. Duas são as linhas celulares obtidas por tumorigênese induzida em camundongos transgênicos: 1) as células GT1 (com os seus subclones GT1-1, GT1-3, GT1-7) e 2) as células GN (com os seus subclones GN10, GN11, NLT). As células GT1 foram derivadas de um tumor hipotalâmico. Pode-se constatar que elas são dotadas de propriedades dos neurônios maduros secretores de GnRH, que completaram o seu trajeto da sua sede de origem, o placóide olfatório, até a sua sede definitiva, o hipotálamo, e já perderam a capacidade de mover-se. Por essas características, as células GT1 são utilizadas sobretudo para o estudo das propriedades secretórias dos neurônios que produzem o GnRH e para identificar os sinais que ali chegam. Pode-se assim evidenciar uma série de receptores, que, ativados pelos seus ligantes (neurotransmissores, hormônios, fatores de crescimento), modulam a síntese e a secreção do GnRH. As células GN foram retiradas de um tumor do bulbo olfatório, portanto, elas são consideradas mais semelhantes aos neurônios imaturos secretores de GnRH que ainda estão desenvolvendo o processo de migração do placóide olfatório até o hipotálamo. Desse modo, tais células são utilizadas sobretudo para identificar e caracterizar os fatores que possam influenciar os processos de migração dos neurônios que produzem o GnRH. Em particular, pode-se constatar que a motilidade dos neurônios secretores desse hormônio é estimulada pela anosmina, a proteína codificada pelo gene KAL1, que, nas suas formas mutantes, ocasiona o hipogonadismo hipogonadotrófico conhecido como a síndrome de Kallmann, por alguns fatores de crescimento (fator de crescimento de fibroblasto, fator de crescimento...

The study of the neurons secreting the gonadotropin releasing hormone (GnRH), the hypothalamic hormone stimulating the release of pituitary gonadotropins, has been potentiated by the development of immortalized cells that specifically synthetize and secrete GnRH. Two cell lines have been obtained by targeted tumorigenesis in transgenic mice: 1) the GT1 cells (with GT1-1, GT1-3 and GT1-7 subclones), and 2) the GN cells (with the GN10, GN11 and NLT subclones). GT1 cells have been obtained from a hypothalamic tumor and exhibit the properties of fully mature GnRH secreting neurons after they reached their final destination in the hypothalamus starting from the olfactory placode. Because of their characteristics GT1 cells have been mainly utilized to investigate the secretory properties of GnRH neurons and to identify the inputs modulating their activity. By this way a consistent number of receptors responding to specific ligands (neurotransmitters, hormones, growth factors) controlling GnRH synthesis and secretion has been identified. GN cells have been derived from a tumor of the olfactory bulb and are considered to replicate the properties of immature GnRH secreting neurons still retaining the capacity of moving. Consequently these cells are used to identify and characterize the factors influencing the migratory process of GnRH neurons from the olfactory placode to the hypothalamus. It has been found that factors stimulating GnRH neuron motility include anosmin, the protein encoded by the KAL1 gene, whose mutations lead to the form of hypogonadotropic hypogonadism known as Kallmann’s syndrome, growth factors such as fibroblast growth factor, hepatocyte growth factor, vascular endothelial growth factor, and cytoskeleton associated proteins (stathmin). On the contrary GABA agonists and glucocorticoids depress GN cells motility. As a whole the findings reported in this review seem particularly important to provide further information on the central...

Reelin provides an inhibitory signal in the migration of gonadotropin-releasing hormone neurons.

Gonadotropin-releasing hormone (GnRH) neurons, a small number of cells scattered in the hypothalamic region of the basal forebrain, play an important role in reproductive function. These cells originate in the olfactory placode and migrate into the basal forebrain in late embryonic life. Here, we show that reelin, which is expressed along the route of the migrating cells, has an inhibitory role in guiding GnRH neurons to the basal forebrain. Only a small (approximately 5%) subpopulation of these neurons expresses one of the reelin receptors (ApoER2/Lrp8), and all GnRH neurons appear to lack the intracellular adaptor protein Dab1, suggesting that the function of reelin is not mediated by the conventional signal transduction pathway. The importance of reelin in the establishment of GnRH neurons in the hypothalamus was confirmed by our finding that the brains of developing and adult reeler mice of both sexes contained a markedly reduced number of these neuroendocrine neurons. Furthermore, the testes of adult males showed dilation of seminiferous tubules and reduction in their density when compared with controls. Mutants lacking the reelin receptors ApoER2 and Vldlr, and scrambler mice lacking Dab1, showed a normal complement of GnRH neurons in the hypothalamus, confirming that the effect of reelin in their migration is independent of Dab1.

Expression and differential effects of the activation of glucocorticoid receptors in mouse gonadotropin-releasing hormone neurons.

Prenatal exposure of rodents to glucocorticoids (Gc) affects the sexual development of the offspring, possibly interfering with the differentiation of the hypothalamic-pituitary-gonadal axis. Glucocorticoid receptors (GR) are present on gonadotropin-releasing hormone (GnRH) neurons in the rat hypothalamus, suggesting a direct effect of Gc in the control of the synthesis and/or release of the hormone. In this study, we demonstrate the colocalization of immunoreactive GR with GnRH in a subpopulation of mouse hypothalamic GnRH neurons, confirming the possible involvement of Gc in mouse GnRH neuronal physiology. Receptor-binding assay, RT-PCR, immunocytochemistry, and immunoblotting experiments carried out in GN11 immortalized GnRH neurons show the presence of GR even in the more immature mouse GnRH neurons and confirm the expression of GR in GT1-7 mature GnRH cells. In GN11 cells, the activation of GR with dexamethasone produces nuclear translocation, but does not lead to the inhibition of GnRH gene expression already reported in GT1-7 cells. Long-term exposure of GN11 cells to dexamethasone induces an epithelial-like phenotype with a reorganization of F-actin in stress fibers. Finally, we found that Gc treatment significantly decreases the migratory activity in vitro and the levels of phosphorylated focal adhesion kinase of GN11 immature neurons. In conclusion, these data indicate that GR are expressed in mouse hypothalamic GnRH neurons in vivo as well as in the immature GN11 GnRH neurons in vitro. Moreover, the effects of the GR activation in GN11 and in GT1-7 cells may be related to the neuronal maturational stage of the two cell lines, suggesting a differential role of Gc in neuronal development.

Hypogonadotropic hypogonadism and peripheral neuropathy in Ebf2-null mice.

Olf/Ebf transcription factors have been implicated in numerous developmental processes, ranging from B-cell development to neuronal differentiation. We describe mice that carry a targeted deletion within the Ebf2 (O/E3) gene. In Ebf2-null mutants, because of defective migration of gonadotropin releasing hormone-synthesizing neurons, formation of the neuroendocrine axis (which is essential for pubertal development) is impaired, leading to secondary hypogonadism. In addition, Ebf2(-/-) peripheral nerves feature defective axon sorting, hypomyelination, segmental dysmyelination and axonal damage, accompanied by a sharp decrease in motor nerve conduction velocity. Ebf2-null mice reveal a novel genetic cause of hypogonadotropic hypogonadism and peripheral neuropathy in the mouse, disclosing an important role for Ebf2 in neuronal migration and nerve development.

Sphingolipid metabolism and caveolin expression in gonadotropin-releasing hormone-expressing GN11 and gonadotropin-releasing hormone-secreting GT1-7 neuronal cells.

In this paper, we show that caveolin-1 is abundantly present in a cell line of immortalized gonadotropin-releasing hormone-expressing neurons (GN11). In contrast to GN11, caveolin is undetectable in a cognate cell line of immortalized gonadotropin-releasing hormone-secreting neurons (GT1-7). These two cell lines are characterized by a radically different sphingolipid metabolism. After incubation in the presence of tracer amount of [1-(3)H]sphingosine, GN11 and GT1-7 neurons incorporated similar amounts of radioactivity. In GT1-7 neurons, [1-(3)H]sphingosine metabolism was markedly oriented toward the biosynthesis of complex sphingolipids. In fact, almost all the radioactivity in the lipid extracts from GT1-7 cells was associated with biosynthetic products (ceramide, sphingomyelin, and glycosphingolipids). In particular glycosphingolipids represented more than 65% of total lipid radioactivity in these cells, and the main glycosphingolipid was GM3 ganglioside (about 47% of total lipid radioactivity). In the case of GN11 neurons, a high portion of [1-(3)H]sphingosine underwent complete degradation, as indicated by the formation of high levels of radioactive phosphatidylethanolamine (about 23% of lipid radioactivity). Moreover, the main complex sphingolipid in GN11 neurons was not a glycolipid, but sphingomyelin (its level in these cells, about 54% of lipid radioactivity, was two-fold higher than in GT1-7). Glycolipids, gangliosides in particular, were present in low amount (9.5% of lipid radioactivity) if compared with the cognate GT1-7 cell line, and GM3 was almost absent in GN11 neurons. Despite the radical differences in ganglioside and caveolin content, from both cell types a membrane fraction similarly enriched in sphingolipids was prepared. In the case of GN11 cells, this fraction was also enriched in caveolin. The presence of caveolin or GM3 may correlate with different functional properties linked to the stage of neuronal maturation, since GN11 and GT1-7 are representative, respectively, of immature, migrating, and differentiated, postmigratory gonadotropin-releasing hormone-positive neurons.