The relationship between pulsatile GnRH secretion and cAMP production in immortalized GnRH neurons

In perifused immortalized GnRH neurons (GT1–7), simultaneous measurements of GnRH and cAMP revealed that the secretory profiles for both GnRH and cAMP are pulsatile. An analysis of GnRH and cAMP pulses in 16 independent experiments revealed that 25% of pulses coincide. Inversion of the peak and nadir levels was found in 33% and random relationship between GnRH and cAMP found in 42% of analyzed pulses. The random relation between GnRH and cAMP pulse resets to synchronous after an inverse relation between pulses occurred during the major GnRH release, indicating that GnRH acts as a switching mechanism to synchronize cAMP and GnRH release in perifused GT1–7 neurons. Activation of GnRH receptors with increasing agonist concentrations caused a biphasic change in cAMP levels. Low nanomolar concentrations increased cAMP production, but at high concentrations the initial increase was followed by a rapid decline to below the basal level. Blockade of the GnRH receptors by peptide and nonpeptide antagonists generated monotonic nonpulsatile increases in both GnRH and cAMP production. These findings indicate that cAMP positively regulates GnRH secretion but does not participate in the mechanism of pulsatile GnRH release.

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Autocrine Regulation of Gonadotropin-Releasing Hormone Secretion in Cultured Hypothalamic Neurons

Endocrinology ◽

10.1210/endo.140.3.6588 ◽

1999 ◽

Vol 140 (3) ◽

pp. 1423-1431 ◽

Cited By ~ 53

Author(s):

Lazar Z. Krsmanovic ◽

Antonio J. Martinez-Fuentes ◽

Krishan K. Arora ◽

Nadia Mores ◽

Carlos E. Navarro ◽

...

Keyword(s):

Hormone Secretion ◽

Receptor Activation ◽

Gnrh Receptor ◽

Hypothalamic Neurons ◽

Gnrh Receptors ◽

Pulsatile Gnrh ◽

Gnrh Neurons ◽

Gnrh Release ◽

Hypothalamic Cells

Abstract Episodic hormone secretion is a characteristic feature of the hypothalamo-pituitary-gonadal system, in which the profile of gonadotropin release from pituitary gonadotrophs reflects the pulsatile secretory activity of GnRH-producing neurons in the hypothalamus. Pulsatile release of GnRH is also evident in vitro during perifusion of immortalized GnRH neurons (GT1–7 cells) and cultured fetal hypothalamic cells, which continue to produce bioactive GnRH for up to 2 months. Such cultures, as well as hypothalamic tissue from adult rats, express GnRH receptors as evidenced by the presence of high-affinity GnRH binding sites and GnRH receptor transcripts. Furthermore, individual GnRH neurons coexpress GnRH and GnRH receptors as revealed by double immunostaining of hypothalamic cultures. In static cultures of hypothalamic neurons and GT1–7 cells, treatment with the GnRH receptor antagonist, [d-pGlu1, d-Phe2, d-Trp3,6]GnRH caused a prominent increase in GnRH release. In perifused hypothalamic cells and GT1–7 cells, treatment with the GnRH receptor agonist, des-Gly10-[d-Ala6]GnRH N-ethylamide, reduced the frequency and increased the amplitude of pulsatile GnRH release, as previously observed in GT1–7 cells. In contrast, exposure to the GnRH antagonist analogs abolished pulsatile secretion and caused a sustained and progressive increase in GnRH release. These findings have demonstrated that GnRH receptors are expressed in hypothalamic GnRH neurons, and that receptor activation is required for pulsatile GnRH release in vitro. The effects of GnRH agonist and antagonist analogs on neuropeptide release are consistent with the operation of an ultrashort-loop autocrine feedback mechanism that exerts both positive and negative actions that are necessary for the integrated control of GnRH secretion from the hypothalamus.

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Evidence That Dynorphin Acts Upon KNDy and GnRH Neurons During GnRH Pulse Termination in the Ewe

Endocrinology ◽

10.1210/en.2018-00435 ◽

2018 ◽

Vol 159 (9) ◽

pp. 3187-3199 ◽

Cited By ~ 14

Author(s):

Peyton W Weems ◽

Lique M Coolen ◽

Stanley M Hileman ◽

Steven Hardy ◽

Rick B McCosh ◽

...

Keyword(s):

Arcuate Nucleus ◽

Third Ventricle ◽

Pulse Generation ◽

G Protein Coupled Receptors ◽

Neurokinin B ◽

Gnrh Secretion ◽

Pulsatile Gnrh ◽

Gnrh Neurons ◽

Kndy Neurons ◽

G Protein Coupled

Abstract A subpopulation of neurons located within the arcuate nucleus, colocalizing kisspeptin, neurokinin B, and dynorphin (Dyn; termed KNDy neurons), represents key mediators of pulsatile GnRH secretion. The KNDy model of GnRH pulse generation proposes that Dyn terminates each pulse. However, it is unknown where and when during a pulse that Dyn is released to inhibit GnRH secretion. Dyn acts via the κ opioid receptor (KOR), and KOR is present in KNDy and GnRH neurons in sheep. KOR, similar to other G protein–coupled receptors, are internalized after exposure to ligand, and thus internalization can be used as a marker of endogenous Dyn release. Thus, we hypothesized that KOR will be internalized at pulse termination in both KNDy and GnRH neurons. To test this hypothesis, GnRH pulses were induced in gonad-intact anestrous ewes by injection of neurokinin B (NKB) into the third ventricle and animals were euthanized at times of either pulse onset or termination. NKB injections produced increased internalization of KOR within KNDy neurons during both pulse onset and termination. In contrast, KOR internalization into GnRH neurons was seen only during pulse termination, and only in GnRH neurons within the mediobasal hypothalamus (MBH). Overall, our results indicate that Dyn is released onto KNDy cells at the time of pulse onset, and continues to be released during the duration of the pulse. In contrast, Dyn is released onto MBH GnRH neurons only at pulse termination and thus actions of Dyn upon KNDy and GnRH cell bodies may be critical for pulse termination.

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60 YEARS OF NEUROENDOCRINOLOGY: The hypothalamo-pituitary–gonadal axis

Journal of Endocrinology ◽

10.1530/joe-15-0113 ◽

2015 ◽

Vol 226 (2) ◽

pp. T41-T54 ◽

Cited By ~ 81

Author(s):

Tony M Plant

Keyword(s):

Gonadal Function ◽

Neuroendocrine Control ◽

Therapeutic Applications ◽

Pulsatile Gnrh ◽

Reproductive Axis ◽

The Future ◽

Gnrh Release ◽

The Relationship ◽

Ovarian Cycles

This review provides an outline of how our understanding of the neuroendocrine control of the hypothalamo-pituitary–gonadal axis has evolved since the publication of Geoffrey Harris' renowned monograph in 1955. Particular attention is directed to the neurobiology underlying pulsatile GnRH release from the hypothalamus, the neuroendocrine control of ovarian cycles, puberty and seasonality of gonadal function, and to ideas that have emerged as a result of examining the relationship between growth and the reproductive axis. The review closes with i) a brief discussion of how knowledge gained as a result of pursing the early hypotheses of Harris has led to major clinical and therapeutic applications, and ii) a personal glimpse into the future of research in this fascinating area of biology.

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Kisspeptin Is Essential for the Full Preovulatory LH Surge and Stimulates GnRH Release from the Isolated Ovine Median Eminence

Endocrinology ◽

10.1210/en.2010-1225 ◽

2011 ◽

Vol 152 (3) ◽

pp. 1001-1012 ◽

Cited By ~ 136

Author(s):

Jeremy T. Smith ◽

Qun Li ◽

Kai Sing Yap ◽

Muhammad Shahab ◽

Antonia K. Roseweir ◽

...

Keyword(s):

Median Eminence ◽

Positive Feedback ◽

Estradiol Benzoate ◽

Portal System ◽

Neuronal Tracing ◽

Lh Surge ◽

External Zone ◽

Gnrh Secretion ◽

Gnrh Neurons ◽

Gnrh Release

Kisspeptins are the product of the Kiss1 gene and potently stimulate GnRH secretion. In sheep, Kiss1 mRNA-expressing cells are found in the arcuate nucleus (ARC) and dorsal-lateral preoptic area and both appear to mediate the positive feedback effect of estradiol to generate the preovulatory GnRH/LH surge. To determine the role of kisspeptin in transmitting estrogen-positive feedback in the hypothalamus, we administered the kisspeptin antagonist p-271 to ewes subjected to an estradiol benzoate-induced LH surge. Kisspeptin antagonist treatment significantly attenuated these LH surges. We further examined the response to kisspeptin treatment prior to the LH surge. Kisspeptin significantly stimulated GnRH secretion into the hypophysial portal system, but the response to kisspeptin was similar in luteal and late-follicular phase ewes. Kiss1r mRNA expression in GnRH neurons was also similar across the estrous cycle. To examine alternative pathways for kisspeptin stimulation of GnRH neurons, we examined the origin of kisspeptin neuronal fibers in the external zone of the median eminence (ME) using neuronal tracing and immunohistochemical techniques. ARC populations of kisspeptin neurons project fibers to the ME. Finally, we showed kisspeptin stimulates GnRH release from ovine ME-cultured explants. This suggests direct kisspeptin to GnRH terminal-to-terminal communication within the ME. Overall, these data indicate an essential role for kisspeptin in receiving stimulatory estrogen signals and generating the full positive feedback GnRH/LH surge. Kisspeptin neurons of the ARC project to the external zone of the ME and kisspeptin acts upon the GnRH fibers at this level.

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Innervation of GnRH Neuron Distal Projections and Activation by Kisspeptin in a New GnRH-Cre Rat Model

Endocrinology ◽

10.1210/endocr/bqaa186 ◽

2020 ◽

Vol 162 (1) ◽

Author(s):

Siew Hoong Yip ◽

Pauline Campos ◽

Xinhuai Liu ◽

Robert Porteous ◽

Allan E Herbison

Keyword(s):

Median Eminence ◽

Pulse Generator ◽

Ectopic Expression ◽

Brain Slices ◽

Gnrh Neuron ◽

External Zone ◽

Pulsatile Gnrh ◽

Gnrh Neurons ◽

Gnrh Release ◽

Presynaptic Nerve Terminals

Abstract The neural mechanisms generating pulsatile GnRH release from the median eminence (ME) remain unclear. Studies undertaken in the mouse demonstrate that GnRH neurons extend projections to the ME that have properties of both dendrites and axons, termed “dendrons,” and that the kisspeptin neuron pulse generator targets these distal dendrons to drive pulsatile GnRH secretion. It presently remains unknown whether the GnRH neuron dendron exists in other species. We report here the generation of a knock-in Gnrh1-Ires-Cre rat line with near-perfect targeting of Cre recombinase to the GnRH neuronal phenotype. More than 90% of adult male and female GnRH neurons express Cre with no ectopic expression. Adeno-associated viruses were used in adult female Gnrh1-Ires-Cre rats to target mCherry or GCAMP6 to rostral preoptic area GnRH neurons. The mCherry tracer revealed the known unipolar and bipolar morphology of GnRH neurons and their principal projection pathways to the external zone of the ME. Synaptophysin-labeling of presynaptic nerve terminals revealed that GnRH neuron distal projections received numerous close appositions as they passed through the arcuate nucleus and into the median eminence. Confocal GCaMP6 imaging in acute horizontal brain slices demonstrated that GnRH neuron distal projections lateral to the median eminence were activated by kisspeptin. These studies indicate the presence of a dendron-like arrangement in the rat with GnRH neuron distal projections receiving synaptic input and responding to kisspeptin.

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Decreased Expression of A-Kinase Anchoring Protein 150 in GT1 Neurons Decreases Neuron Excitability and Frequency of Intrinsic Gonadotropin-Releasing Hormone Pulses

Endocrinology ◽

10.1210/en.2009-0894 ◽

2010 ◽

Vol 151 (1) ◽

pp. 281-290 ◽

Cited By ~ 4

Author(s):

Qiumei Chen ◽

Richard I. Weiner ◽

Brigitte E. Blackman

Keyword(s):

Intracellular Camp ◽

Mrna Levels ◽

Gnrh Secretion ◽

Protein Levels ◽

Pulsatile Gnrh ◽

Neuron Excitability ◽

Gnrh Neurons ◽

Anchoring Protein ◽

Infected Cells

Abstract The frequency of intrinsic pulsatile GnRH secretion from endogenous GnRH neurons and GT1 GnRH cell lines is stimulated by increased intracellular cAMP levels. The downstream molecules comprising the cAMP signaling pathway are organized in microdomains by a family of scaffolding proteins, A-kinase anchoring proteins (AKAPs). These molecules tether protein kinase A, cAMP-specific phosphodiesterases, phosphatases to known substrates. In neurons AKAP150 organizes many of the signaling molecules known to regulate the excitability and intrinsic pulsatile activity of GnRH neurons. AKAP150 was expressed in both the GT1-1 and GT1-7 cells. We determined the role of AKAP150 in coordinating GT1-1 cell excitability and intrinsic GnRH pulsatile secretion by lowering AKAP150 levels with a small interfering RNA (siRNA) adenovirus construct to AKAP150 (Ad-AKAP150-siRNA). Infection with Ad-AKAP150-siRNA specifically decreased AKAP150 mRNA levels by 74% and protein levels by 53% relative to uninfected cells or cells infected with a luciferase control adenovirus siRNA vector. In GT1 cells, spontaneous Ca2+ oscillations, an index of neuron excitability, are stimulated by increased levels of intracellular cAMP and lowered by decreased levels. The frequency of spontaneous Ca2+ oscillations in Ad-AKAP150-siRNA-treated GT1-1 cells decreased by 47.2% relative to controls. A dramatic decrease in the number of spontaneous GnRH pulses was also observed after infection with Ad-AKAP150-siRNA. The interpulse interval increased to 143 ± 20.25 min in Ad-AKAP150-siRNA infected cells from 32.2 ± 7.3 min in luciferase control adenovirus siRNA vector-infected cells. These data demonstrate an important role of AKAP150 in coordinating signaling events regulating the frequency of intrinsic pulsatile GnRH secretion.

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Converse Regulatory Functions of Estrogen Receptor-α and -β Subtypes Expressed in Hypothalamic Gonadotropin-Releasing Hormone Neurons

Molecular Endocrinology ◽

10.1210/me.2008-0192 ◽

2008 ◽

Vol 22 (10) ◽

pp. 2250-2259 ◽

Cited By ~ 42

Author(s):

Lian Hu ◽

Robert L. Gustofson ◽

Hao Feng ◽

Po Ki Leung ◽

Nadia Mores ◽

...

Keyword(s):

Estrogen Receptor Α ◽

Female Rats ◽

Neuronal Membrane ◽

Camp Production ◽

Membrane Excitability ◽

Gnrh Secretion ◽

Adult Rat ◽

Gnrh Neurons ◽

Spontaneous Action ◽

Regulatory Functions

Abstract Estradiol (E2) acts as a potent feedback molecule between the ovary and hypothalamic GnRH neurons, and exerts both positive and negative regulatory actions on GnRH synthesis and secretion. However, the extent to which these actions are mediated by estrogen receptors (ERs) expressed in GnRH neurons has been controversial. In this study, Single-cell RT-PCR revealed the expression of both ERα and ERβ isoforms in cultured fetal and adult rat hypothalamic GnRH neurons. Both ERα and ERβ or individual ERs were expressed in 94% of cultured fetal GnRH neurons. In adult female rats at diestrus, 68% of GnRH neurons expressed ERs, followed by 54% in estrus and 19% in proestrus. Expression of individual ERs was found in 24% of adult male GnRH neurons. ERα exerted marked Gi-mediated inhibitory effects on spontaneous action potential (AP) firing, cAMP production, and pulsatile GnRH secretion, indicating its capacity for negative regulation of GnRH neuronal function. In contrast, increased E2 concentration and ERβ agonists increase the rate of AP firing, GnRH secretion, and cAMP production, consistent with ERβ-dependent positive regulation of GnRH secretion. Consonant with the coupling of ERα to pertussis toxin-sensitive Gi/o proteins, E2 also activates G protein-activated inwardly rectifying potassium channels, decreasing membrane excitability and slowing the firing of spontaneous APs in hypothalamic GnRH neurons. These findings demonstrate that the dual actions of E2 on GnRH neuronal membrane excitability, cAMP production, and GnRH secretion are mediated by the dose-dependent activation of ERα and ERβ expressed in hypothalamic GnRH neurons.

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Pulsatile GnRH secretion from primary cultures of sheep olfactory placode explants

Reproduction ◽

10.1530/reprod/120.2.391 ◽

2000 ◽

pp. 391-396 ◽

Cited By ~ 11

Author(s):

AH Duittoz ◽

M Batailler

Keyword(s):

Culture Medium ◽

Primary Cultures ◽

Pulsatile Secretion ◽

Olfactory Placode ◽

Gnrh Secretion ◽

Individual Culture ◽

Pulsatile Gnrh

The aim of this study was to investigate the development of pulsatile GnRH secretion by GnRH neurones in primary cultures of olfactory placodes from ovine embryos. Culture medium was collected every 10 min for 8 h to detect pulsatile secretion. In the first experiment, pulsatile secretion was studied in two different sets of cultures after 17 and 24 days in vitro. In the second experiment, a set of cultures was tested after 10, 17 and 24 days in vitro to investigate the development of pulsatile GnRH secretion in each individual culture. This study demonstrated that (i) primary cultures of GnRH neurones from olfactory explants secreted GnRH in a pulsatile manner and that the frequency and mean interpulse duration were similar to those reported in castrated ewes, and (ii) pulsatile secretion was not present at the beginning of the culture but was observed between 17 and 24 days in vitro, indicating the maturation of individual neurones and the development of their synchronization.

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Potential Role of Inflammation in Associations between Particulate Matter and Heart Failure

Current Pharmaceutical Design ◽

10.2174/1381612824666180110150550 ◽

2018 ◽

Vol 24 (3) ◽

pp. 341-358 ◽

Cited By ~ 7

Author(s):

Xiaotong Ji ◽

Yingying Zhang ◽

Guangke Li ◽

Nan Sang

Keyword(s):

Heart Failure ◽

Particulate Matter ◽

Inflammatory Response ◽

Heart Diseases ◽

Experimental Studies ◽

Epidemiological Studies ◽

Inflammatory Mechanism ◽

High Concentrations ◽

Future Work ◽

The Relationship

Recently, numerous studies have found that particulate matter (PM) exposure is correlated with increased hospitalization and mortality from heart failure (HF). In addition to problems with circulation, HF patients often display high expression of cytokines in the failing heart. Thus, as a recurring heart problem, HF is thought to be a disorder characterized in part by the inflammatory response. In this review, we intend to discuss the relationship between PM exposure and HF that is based on inflammatory mechanism and to provide a comprehensive, updated evaluation of the related studies. Epidemiological studies on PM-induced heart diseases are focused on high concentrations of PM, high pollutant load exposure in winter, or susceptible groups with heart diseases, etc. Furthermore, it appears that the relationship between fine or ultrafine PM and HF is stronger than that between HF and coarse PM. However, fewer studies paid attention to PM components. As for experimental studies, it is worth noting that coarse PM may indirectly promote the inflammatory response in the heart through systematic circulation of cytokines produced primarily in the lungs, while ultrafine PM and its components can enter circulation and further induce inflammation directly in the heart. In terms of PM exposure and enhanced inflammation during the pathogenesis of HF, this article reviews the following mechanisms: hemodynamics, oxidative stress, Toll-like receptors (TLRs) and epigenetic regulation. However, many problems are still unsolved, and future work will be needed to clarify the complex biologic mechanisms and to identify the specific components of PM responsible for adverse effects on heart health.

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Effect of Maritime Traffic on Water Quality Parameters in Santa Marta, Colombia

Journal of Marine Science and Engineering ◽

10.3390/jmse9050474 ◽

2021 ◽

Vol 9 (5) ◽

pp. 474

Author(s):

René Rodríguez-Grimón ◽

Nestor Hernando Campos ◽

Ítalo Braga Castro

Keyword(s):

Water Quality ◽

Petroleum Hydrocarbons ◽

Quality Parameters ◽

Water Quality Parameters ◽

Maritime Traffic ◽

Natural Park ◽

High Concentrations ◽

The Impact ◽

The Relationship ◽

Multiple Chemical

Since 2013, there has been an increase (>23%) in naval traffic using maritime routes and ports on the coastal fringe of Santa Marta, Colombia. Of major concern, and described by several studies, is the relationship between maritime traffic and coastal contamination. This study proposed a maritime traffic indicator considering the simultaneous effects of several relevant measurements of water quality parameters to estimate the impact of naval activity. The approach involved developing a model including the number of vessels, hull length, and permanence time in berths. In addition, water quality variables, considering climatic seasons, were used to verify association with maritime traffic and touristic activities. The high concentrations of total coliforms (TC) and dissolved/dispersed petroleum hydrocarbons in chrysene equivalents (DDPH) reported by the International Marina of Santa Marta (SM) were affected by the local anthropic activities, including tourism, naval traffic, and urban wastewater discharges. Moreover, our results suggest the occurrence of multiple chemical impacts within Tayrona National Natural Park (PNNT) affecting conservation goals. The estimation of the maritime traffic indicator proposed in this study may be an easy and more complete tool for future studies evaluating the impact of naval activities on environmental quality.

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