Discovery and Evolutionary History of Gonadotrophin‐Inhibitory Hormone and Kisspeptin: New Key Neuropeptides Controlling Reproduction

Gonadotrophin‐releasing hormone (GnRH) is the primary hypothalamic factor responsible for the control of gonadotrophin secretion in vertebrates. However, within the last decade, two other hypothalamic neuropeptides have been found to play key roles in the control of reproductive functions: gonadotrophin‐inhibitory hormone (GnIH) and kisspeptin. In 2000, we discovered GnIH in the quail hypothalamus. GnIH inhibits gonadotrophin synthesis and release in birds through actions on GnRH neurones and gonadotrophs, mediated via GPR147. Subsequently, GnIH orthologues were identified in other vertebrate species from fish to humans. As in birds, mammalian and fish GnIH orthologues inhibit gonadotrophin release, indicating a conserved role for this neuropeptide in the control of the hypothalamic‐pituitary‐gonadal axis across species. Subsequent to the discovery of GnIH, kisspeptin, encoded by the KiSS‐1 gene, was discovered in mammals. By contrast to GnIH, kisspeptin has a direct stimulatory effect on GnRH neurones via GPR54. GPR54 is also expressed in pituitary cells, but whether gonadotrophs are targets for kisspeptin remains unresolved. The KiSS‐1 gene is also highly conserved and has been identified in mammals, amphibians and fish. We have recently found a second isoform of KiSS‐1, designated KiSS‐2, in several vertebrates, but not birds, rodents or primates. In this review, we highlight the discovery, mechanisms of action, and functional significance of these two chief regulators of the reproductive axis.     

Diurnal and Circadian Oscillations in Expression of Kisspeptin,Kisspeptin Receptor and Gonadotrophin‐Releasing Hormone 2 Genes in the Grass Puffer,A Semilunar‐Synchronised Spawner

In seasonally breeding animals, the circadian and photoperiodic regulation of neuroendocrine system is important for precisely‐timed reproduction. Kisspeptin, encoded by the Kiss1 gene, acts as a principal positive regulator of the reproductive axis by stimulating gonadotrophin‐releasing hormone (GnRH) neurone activity in vertebrates. However, the precise mechanisms underlying the cyclic regulation of the kisspeptin neuroendocrine system remain largely unknown. The grass puffer, Takifugu niphobles, exhibits a unique spawning rhythm: spawning occurs 1.5–2 h before high tide on the day of spring tide every 2 weeks, and the spawning rhythm is connected to circadian and lunar‐/tide‐related clock mechanisms. The grass puffer has only one kisspeptin gene (kiss2), which is expressed in a single neural population in the preoptic area (POA), and has one kisspeptin receptor gene (kiss2r), which is expressed in the POA and the nucleus dorsomedialis thalami. Both kiss2 and kiss2r show diurnal variations in expression levels, with a peak at Zeitgeber time (ZT) 6 (middle of day time) under the light/dark conditions. They also show circadian expression with a peak at circadian time 15 (beginning of subjective night‐time) under constant darkness. The synchronous and diurnal oscillations of kiss2 and kiss2r expression suggest that the action of Kiss2 in the diencephalon is highly dependent on time. Moreover, midbrain GnRH2 gene (gnrh2) but not GnRH1 or GnRH3 genes show a unique semidiurnal oscillation with two peaks at ZT6 and ZT18 within a day. The cyclic expression of kiss2, kiss2r and gnrh2 may be important in the control of the precisely‐timed diurnal and semilunar spawning rhythm of the grass puffer, possibly through the circadian clock and melatonin, which may transmit the photoperiodic information of daylight and moonlight to the reproductive neuroendocrine centre in the hypothalamus.     

Differential response patterns of kisspeptin and RFamide‐related peptide to photoperiod and sex steroid feedback in the Djungarian hamster (Phodopus sungorus)

Many animals synchronise their reproductive activity with the seasons to optimise the survival of their offspring. This synchronisation involves switching on and off their gonadotrophic axis. Ever since their discovery as key regulators of gonadotrophin‐releasing hormone (GnRH) neurones, the hypothalamic RF‐amide peptides kisspeptin and RFamide‐related peptide (RFRP) have been a major focus of research on the seasonal regulation of the gonadotrophic axis. In the present study, we investigated the regulation of both neuropeptides in the Djungarian hamster, a major animal model for the study of seasonal reproduction. During the long‐day breeding period, kisspeptin neurones in the anteroventral periventricular area are solely controlled by a positive sex steroid feedback and, in the arcuate nucleus, they are subject to a very strong negative sex steroid feedback associated with a minor photoperiodic effect. During short‐day sexual quiescence, the disappearance of this hormonal feedback leads to high levels of kisspeptin in arcuate neurones. Notably, chronic central administration of kisspeptin is able to over‐ride the photoperiodic inhibition of the gonadotrophic axis and reactivate the reproductive function. Therefore, our data suggest that kisspeptin secretion by arcuate neurones during sexual quiescence is inhibited by mechanisms upstream of kisspeptin neurones. RFRP expression is solely controlled by photoperiod, being strongly reduced in short days independently of the sex steroid feedback. Thus, kisspeptin and RFRP display contrasting patterns of expression and regulation. Upstream mechanisms controlling these neurones should be the focus of further studies on the roles of these RFamide neuropeptides in the seasonal control of reproduction.     

Effect of Deletion of Ghrelin‐O‐Acyltransferase on the Pulsatile Release of Growth Hormone in Mice

Ghrelin, a gut hormone originating from the post‐translational cleavage of preproghrelin, is the endogenous ligand of growth hormone secretagogue receptor 1a (GHS‐R1a). Within the growth hormone (GH) axis, the biological activity of ghrelin requires octanoylation by ghrelin‐O‐acyltransferase (GOAT), conferring selective binding to the GHS‐R1a receptor via acylated ghrelin. Complete loss of preproghrelin‐derived signalling (through deletion of the Ghrl gene) contributes to a decline in peak GH release; however, the selective contribution of endogenous acyl‐ghrelin to pulsatile GH release remains to be established. We assessed the pulsatile release of GH in ad lib. fed male germline goat^?/? mice, extending measures to include mRNA for key hypothalamic regulators of GH release, and peripheral factors that are modulated relative to GH release. The amount of GH released was reduced in young goat^?/? mice compared to age‐matched wild‐type mice, whereas pulse frequency and irregularity increased. Altered GH release did not coincide with alterations in hypothalamic Ghrh, Srif, Npy or Ghsr mRNA expression, or pituitary GH content, suggesting that loss of Goat does not compromise canonical mechanisms that contribute to pituitary GH production and release. Although loss of Goat resulted in an irregular pattern of GH release (characterised by an increase in the number of GH pulses observed during extended secretory events), this did not contribute to a change in the expression of sexually dimorphic GH‐dependent liver genes. Of interest, circulating levels of insulin‐like growth factor (IGF)‐1 were elevated in goat^?/? mice. This rise in circulating levels of IGF‐1 was correlated with an increase in GH pulse frequency, suggesting that sustained or increased IGF‐1 release in goat^?/? mice may occur in response to altered GH release patterning. Our observations demonstrate that germline loss of Goat alters GH release and patterning. Although the biological relevance of altered GH secretory patterning remains unclear, we propose that this may contribute to sustained IGF‐1 release and growth in goat^?/? mice.     

Maturation of Sleep–Wake Gonadotrophin‐Releasing Hormone Secretion Across Puberty in Girls: Potential Mechanisms and Relevance to the Pathogenesis of Polycystic Ovary Syndrome

Neuroendocrine mechanisms underlying the progression of sleep–wake gonadotrophin‐releasing hormone (GnRH) pulse secretion across puberty have remained enigmatic. Here, the changes of sleep–wake luteinising hormone (LH) (and, by inference, GnRH) pulse secretion across puberty in normal girls are reviewed, primarily focusing on available human data. It is suggested that the primary control of GnRH pulse frequency changes across puberty, with sex steroid feedback exerting minimal control during childhood, but primary control during adulthood. A working model is proposed regarding how such a transfer of GnRH pulse frequency control may partly account for the prominent day–night differences of GnRH pulse frequency characteristic of puberty. How this model may be relevant to the genesis of abnormal GnRH secretion in peripubertal girls with hyperandrogenaemia is then described.     

Possible Involvement of Microglia Containing Cyclooxygenase‐1 in the Accumulation of Gonadotrophin‐Releasing Hormone in the Preoptic Area in Female Rats

Prostaglandins (PGs), especially PGE₂, are involved in the hypothalamic control of gonadotrophin‐releasing hormone (GnRH) release, acting at least in part on the terminal of GnRH axons in the median eminence. The present study aimed: (i) to clarify the role of PG(s) in regulating GnRH cell function at the level of the perikarya in the preoptic area; (ii) to determine the cyclooxygenase (COX) isozyme responsible for producing PG(s) that regulates GnRH perikarya; and (iii) to identify cell types that contain the responsible COX isozyme in female rats. A surge of luteinising hormone (LH) secretion was induced by oestrogen and progesterone in ovariectomised rats. Treatment of the rat before the LH surge with indomethacin, a nonselective COX inhibitor, or NS‐398, a selective COX‐2 inhibitor, did not interfere with the surge. However, treatment with indomethacin or flurbiprofen, a selective COX‐1 inhibitor, significantly reduced the number of GnRH‐immunoreactive cells in the preoptic area at the time of peak LH secretion during the surge. NS‐398 did not affect the GnRH immunoreactivity. Double‐labelled immunofluorescent histochemistry revealed COX‐1 immunoreactivity in the vicinity of, but not within, GnRH containing neurones in the preoptic area. COX‐2 immunoreactivity was not found in the same area. The COX‐1 immunoreactivity was almost entirely localised in microglia in the preoptic area, but not in neurones or astrocytes. These results suggest that microglia in the preoptic area containing COX‐1 are responsible for producing PG(s), which, in turn, facilitates the accumulation of GnRH during the gonadotrophin surge in female rats.     

Encoding and decoding mechanisms of pulsatile hormone secretion

Ultradian pulsatile hormone secretion underlies the activity of most neuroendocrine systems, including the hypothalamic‐pituitary adrenal (HPA) and gonadal (HPG) axes, and this pulsatile mode of signalling permits the encoding of information through both amplitude and frequency modulation. In the HPA axis, glucocorticoid pulse amplitude increases in anticipation of waking, and, in the HPG axis, changing gonadotrophin‐releasing hormone pulse frequency is the primary means by which the body alters its reproductive status during development (i.e. puberty). The prevalence of hormone pulsatility raises two crucial questions: how are ultradian pulses encoded (or generated) by these systems, and how are these pulses decoded (or interpreted) at their target sites? We have looked at mechanisms within the HPA axis responsible for encoding the pulsatile mode of glucocorticoid signalling that we observe in vivo. We review evidence regarding the ‘hypothalamic pulse generator’ hypothesis, and describe an alternative model for pulse generation, which involves steroid feedback‐dependent endogenous rhythmic activity throughout the HPA axis. We consider the decoding of hormone pulsatility by taking the HPG axis as a model system and focussing on molecular mechanisms of frequency decoding by pituitary gonadotrophs.     

Oestrogen-dependent suppression of pulsatile luteinising hormone secretion and kiss1 mRNA expression in the arcuate nucleus during late lactation in rats

Follicular development and ovulation are strongly suppressed during lactation in mammals via a profound suppression of gonadotrophin secretion. The present study aimed to examine the role of oestrogen feedback action in suppressing luteinising hormone (LH) secretion and hypothalamic kisspeptin expression during the latter half of lactation. Plasma LH concentrations kept at low levels throughout the lactating period in intact and oestrogen‐replaced ovariectomised (OVX) lactating rats, whereas plasma LH concentrations gradually elevated from day 10 postpartum in lactating OVX rats. OVX lactating rats showed frequent LH pulses at late lactation, although the LH pulses were significantly inhibited by an oestrogen replacement, which is much less effective on LH release in nonlactating rats. Oestrogen replacement in lactating OVX rats significantly reduced the number of Kiss1 mRNA‐expressing cells in the arcuate nucleus (ARC) at late lactation, although the same oestrogen treatment did not affect the number of Kiss1‐expressing cells in nonlactating controls. Exogenous kisspeptin challenge (0.2 nmol) into the third cerebroventricle significantly increased LH secretion in lactating OVX, lactating OVX + subcutaneous 17β‐oestradiol and intact lactating rats at day 16 postpartum. These results suggest that LH pulse suppression during late lactation could be a result of the enhanced oestrogen‐dependent suppression of ARC kisspeptin expression.     

Role of Pituitary Adenylate Cyclase‐Activating Polypeptide in Modulating Hypothalamus‐Pituitary Neuroendocrine Functions in Mouse Cell Models

Pituitary adenylate cyclase‐activating polypeptide (PACAP) was originally identified as a hypothalamic activator of cyclic adenosine monophosphate production in pituitary cells. PACAP and its receptor are expressed not only in the central nervous system, but also in peripheral organs, and function to stimulate pituitary hormone synthesis and secretion as both a hypothalamic‐pituitary‐releasing factor and an autocrine‐paracrine factor within the pituitary. PACAP stimulates the expression of the gonadotrophin α, luteinising hormone (LH) β and follicle‐stimulating hormone (FSH) β subunits, as well as the gonadotrophin‐releasing hormone (GnRH) receptor and its own PACAP type I receptor (PAC1R) in gonadotrophin‐secreting pituitary cells. In turn, GnRH, which is known to be a crucial component of gonadotrophin secretion, stimulates the expression of PACAP and PAC1R in gonadotrophs. In addition, PAC1R and PACAP modulate the functions of GnRH‐producing neurones in the hypothalamus. This review summarises the current understanding of the possible roles of PACAP and PAC1R in modulating hypothalamus and pituitary neuroendocrine cells in the mouse models.     

Molecular Analysis of the Koala Reproductive Hormones and Their Receptors: Gonadotrophin‐Releasing Hormone (GnRH), Follicle‐Stimulating Hormone β and Luteinising Hormone β with Localisation of GnRH

During evolution, reproductive hormones and their receptors in the brain‐pituitary‐gonadal axis have been altered by genetic mechanisms. To understand how the neuroendocrine control of reproduction evolved in mammals, it is important to examine marsupials, the closest group to placental mammals. We hypothesised that at least some of the hormones and receptors found in placental mammals would be present in koala, a marsupial. We examined the expression of koala mRNA for the reproductive molecules. Koala cDNAs were cloned from brain for gonadotrophin‐releasing hormones (GnRH1 and GnRH2) or from pituitary for GnRH receptors, types I and II, follicle‐stimulating hormone (FSH)β and luteinising hormone (LH)β, and from gonads for FSH and LH receptors. Deduced proteins were compared by sequence alignment and phylogenetic analysis with those of other vertebrates. In conclusion, the koala expressed mRNA for these eight putative reproductive molecules, whereas at least one of these molecules is missing in some species in the amniote lineage, including humans. In addition, GnRH1 and 2 are shown by immunohistochemistry to be expressed as proteins in the brain.     

Seasonal Effect of Gonadotrophin Inhibitory Hormone on Gonadotrophin‐Releasing Hormone‐induced Gonadotroph Functions in the Goldfish Pituitary

We have shown that native goldfish gonadotrophin inhibitory hormone (gGnIH) differentially regulates luteinsing hormone (LH)‐β and follicle‐stimulating hormone (FSH)‐β expression. To further understand the functions of gGnIH, we examined its interactions with two native goldfish gonadotrophin‐releasing hormones, salmon gonadotrophin‐releasing hormone (sGnRH) and chicken (c)GnRH‐II in vivo and in vitro. Intraperitoneal injections of gGnIH alone reduced serum LH levels in fish in early and mid gonadal recrudescence; this inhibition was also seen in fish co‐injected with either sGnRH or cGnRH‐II during early recrudescence. Injection of gGnIH alone elevated pituitary LH‐β and FSH‐β mRNA levels at early and mid recrudescence, and FSH‐β mRNA at late recrudescence. Co‐injection of gGnIH attenuated the stimulatory influences of sGnRH on LH‐β in early recrudescence, and LH‐β and FSH‐β mRNA levels in mid and late recrudescence, as well as the cGnRH‐II‐elicited increase in LH‐β, but not FSH‐β, mRNA expression at mid and late recrudescence. sGnRH and cGnRH‐II injection increased pituitary gGnIH‐R mRNA expression in mid and late recrudescence but gGnIH reduced gGnIH‐R mRNA levels in late recrudescence. gGnIH did not affect basal LH release from perifused pituitary cells and continual exposure to gGnIH did not alter the LH responses to acute applications of GnRH. However, a short 5‐min GnIH treatment in the middle of a 60‐min GnRH perifusion selectively reduced the cGnRH‐II‐induced release of LH. These novel results indicate that, in goldfish, gGnIH and GnRH modulate pituitary GnIH‐R expression and gGnIH differentially affects sGnRH and cGnRH‐II regulation of LH secretion and gonadotrophin subunit mRNA levels. Furthermore, these actions are manifested in a reproductive stage‐dependent manner.     

Prenatal Exposure to Vinclozolin Disrupts Selective Aspects of the Gonadotrophin‐Releasing Hormone Neuronal System of the Rabbit

Developmental exposure to the agricultural fungicide vinclozolin can impair reproductive function in male rabbits and was previously found to decrease the number of immunoreactive‐gonadotrophin‐releasing hormone (GnRH) neurones in the region of the organum vasculosum of the lamina terminalis and rostral preoptic area by postnatal week (PNW) 6. In the present study, in an aim to further examine the disruption of GnRH neurones by foetal vinclozolin exposure, pregnant rabbits were dosed orally with vinclozolin, flutamide or carrot paste vehicle for the last 2 weeks of gestation. Offspring were euthanised at birth (males and females), PNW 6 (females), PNW 26 (adult males) or PNW 30 (adult females) of age. At birth and in adults, brains were sectioned and processed for immunoreactive GnRH. The numbers of immunoreactive GnRH neuronal perikarya were significantly decreased in vinclozolin‐treated rabbits at birth and in adult littermates. By contrast, there was an increase in GnRH immunoreactivity in the terminals in the region of the median eminence. Analysis of PNW 6 female brains by radioimmunoassay revealed a two‐fold increase in GnRH peptide content in the mediobasal hypothalamus in vinclozolin‐treated rabbits. This finding was complemented by immunofluorescence analyses, which revealed a 2.8‐fold increase in GnRH immunoreactivity in the median eminence of vinclozolin compared to vehicle‐treated females at PNW 30. However, there was no difference between treatment groups in the measures of reproduction that were evaluated: ejaculation latency, conception rates or litter size. These results indicate that sub‐acute, prenatal vinclozolin treatment is sufficient to create perdurable alterations in the GnRH neuronal network that forms an important input into the reproductive axis. Finally, the effect of vinclozolin on the GnRH neuronal network was not comparable to that of flutamide, suggesting that vinclozolin was not acting through anti‐androgenic mechanisms.     

Regulation of hypothalamic corticotropin‐releasing hormone neurone excitability by oxytocin

Oxytocin (OT) is a neuropeptide that exerts multiple actions throughout the brain and periphery. Within the brain, OT regulates diverse neural populations, including neural networks controlling responses to stress. Local release of OT within the paraventricular nucleus (PVN) of the hypothalamus has been suggested to regulate stress responses by modulating the excitability of neighbouring corticotropin‐releasing hormone (CRH) neurones. However, the mechanisms by which OT regulates CRH neurone excitability are unclear. In the present study, we investigated the morphological relationship between OT and CRH neurones and determined the effects of OT on CRH neurone excitability. Morphological analysis revealed that the processes of OT and CRH neurones were highly intermingled within the PVN, possibly allowing for local cell‐to‐cell cross‐talk. Whole‐cell patch‐clamp recordings from CRH neurones were used to study the impact of OT on postsynaptic excitability and synaptic innervation. Bath‐applied OT did not alter CRH neurone holding current, spiking output or any action potential parameters. Recordings of evoked excitatory and inhibitory postsynaptic currents (EPSCs/IPSCs) revealed no net effect of OT on current amplitude; however, subgroups of CRH neurones appeared to respond differentially to OT. Analysis of spontaneous EPSC events uncovered a significant reduction in spontaneous EPSC frequency but no change in spontaneous EPSC amplitude in response to OT. Together, these data demonstrate that OT exerts a subtle modulation of synaptic transmission onto CRH neurones providing one potential mechanism by which OT could suppress CRH neurone excitability and stress axis activity.     

Effects of Chronic NMDA‐NR2b Inhibition in the Median Eminence of the Reproductive Senescent Female Rat

Gonadotrophin‐releasing hormone (GnRH) neurones of the hypothalamic‐pituitary‐gonadal (HPG) axis drive reproductive function and undergo age‐related decreases in activation during the transition to reproductive senescence. Decreased GnRH secretion from the median eminence (ME) partially arises from attenuated glutamatergic signalling via the NMDA receptor (NMDAR) and may be a result of changing NMDAR stoichiometry to favour NR2b over NR2a subunit expression with ageing. We have previously shown that the systemic inhibition of NR2b‐containing receptors with ifenprodil, an NR2b‐specific antagonist, stimulates parameters of luteinising hormone (used as a proxy for GnRH) release in both young and middle‐aged females. In the present study, we chronically administered ifenprodil, an NR2b‐specific antagonist, at the site of GnRH terminals in the ME or at GnRH perikarya in the preoptic area, in reproductively senescent middle‐aged female rats, aiming to determine whether NR2b antagonism could restore aspects of reproductive functionality. Effects on oestrous cyclicity, serum hormones, and protein expression of GnRH, NR2b and phosphorylated NR2b (Tyr‐1472) in the ME were measured. Chronic ifenprodil treatment in the ME (but not the preoptic area) altered oestrous cyclicity by increasing the percentage of days spent in pro‐oestrus. This was accompanied by increased GnRH fluorescence intensity in the external ME zone and a greater proportion of GnRH terminals that co‐labelled with pNR2b with treatment. We also observed changes in the relationships between protein immunofluorescence, serum hormone levels and other aspects of reproductive physiology in acyclic females, as revealed by bionetwork analysis. Together, these data support the hypothesis that NMDAR‐NR2b expression and phosphorylation state play a role in reproductive senescence and highlight the ME as a major player in reproductive ageing.     

Oestrogen induces rhythmic expression of the Kisspeptin-1 receptor GPR54 in hypothalamic gonadotrophin-releasing hormone-secreting GT1-7 cells

Oestrogen-stimulated preovulatory gonadotrophin surges are temporally regulated in a way that remains not fully understood. Mammalian ovulation requires surges of gonadotrophin-releasing hormone (GnRH), released from specialised neurones in the hypothalamus. Surge regulation is mediated by ovarian oestrogen (17 β-oestradiol; E(2) ) feedback-acting as a negative signal until the early afternoon of the pro-oestrous phase, at which point it stimulates robust increases in GnRH release. Multiple lines of evidence suggest a role for the circadian clock in surge generation, although the presence of endogenous oscillators in several neuronal populations throughout the mediobasal hypothalamus complicates an elucidation of the underlying mechanisms of circadian regulation. In the present study, we propose that endogenous oscillators within GnRH neurones are modulated by oestrogen to elicit GnRH surge secretion. One mechanism by which this may occur is through the up-regulation of receptors of known stimulators of GnRH, such as kisspeptin's cognate receptor, GPR54. Through analysis of mRNA and protein abundance patterns, we found that high levels of E(2) elicit circadian expression profiles of GPR54 in vitro, and that disruption of endogenous GnRH oscillators of the clock dampens this effect. Additionally, although kisspeptin administration to GT1-7 cells does not result in surge-level secretion, we observed increased GnRH secretion from GT1-7 cells treated with positive feedback levels of E(2) . These results in this in vitro neuronal model system suggest a possible mechanism whereby receptor expression levels, and thus GnRH sensitivity to kisspeptin, may change dramatically over the pro-oestrous day. In this way, elevated ovarian E(2) may increase kisspeptidergic tone at the same time as increasing GnRH neuronal sensitivity to this neuropeptide for maximal surge release.     

Gonadotrophin‐Inhibitory Hormone in the Cichlid Fish Cichlasoma dimerus: Structure,Brain Distribution and Differential Effects on the Secretion of Gonadotrophins and Growth Hormone

The role of gonadotrophin‐inhibitory hormone (GnIH) in the inhibition of the reproductive axis has been well‐established in birds and mammals. However, its role in other vertebrates, such as the teleost fish, remains controversial. In this context, the present study aimed to evaluate whether GnIH modulates the release of gonadotrophins and growth hormone (GH) in the cichlid fish Cichlasoma dimerus. First, we partially sequenced the precursor polypeptide for GnIH and identified three putative GnIH peptides. Next, we analysed the expression of this precursor polypeptide via a polymerase chain reaction in the reproductive axis of both sexes. We found a high expression of the polypeptide in the hypothalamus and gonads of males. Immunocytochemistry allowed the observation of GnIH‐immunoreactive somata in the nucleus posterioris periventricularis and the nucleus olfacto‐retinalis, with no differences between the sexes. GnIH‐immunoreactive fibres were present in all brain regions, with a high density in the nucleus lateralis tuberis and at both sides of the third ventricle. Finally, we performed in vitro studies on intact pituitary cultures to evaluate the effect of two doses (10^?6 m and 10^?8 m ) of synthetic C. dimerus (cd‐) LPQRFa‐1 and LPQRFa‐2 on the release of gonadotrophins and GH. We observed that cd‐LPQRFa‐1 decreased β‐luteinising hormone (LH) and β‐follicle‐stimulating hormone (FSH) and also increased GH release to the culture medium. The release of β‐FSH was increased only when it was stimulated with the higher cd‐LPQRFa‐2 dose. The results of the present study indicate that cd‐LPQRFa‐1, the cichlid fish GnIH, inhibits β‐LH and β‐FSH release and stimulates GH release in intact pituitary cultures of C. dimerus. The results also show that cd‐LPQRF‐2 could act as an β‐FSH‐releasing factor in this fish species.     

Interleukin‐6 increases intracellular Ca2+ concentration and induces catecholamine secretion in rat carotid body glomus cells

Although abundant evidence indicates mutual regulation between the immune and the central nervous systems, how the immune signals are transmitted to the brain is still an unresolved question. In a previous study we found strong expression of proinflammatory cytokine receptors, including interleukin (IL)‐1 receptor I and IL‐6 receptor α in the rat carotid body (CB), a well‐known arterial chemoreceptor that senses a variety of chemostimuli in the arterial blood. We demonstrated that IL‐1 stimulation increases intracellular calcium ([Ca²⁺]_i) in CB glomus cells, releases ATP, and increases the discharge rate in carotid sinus nerve. To explore the effect of IL‐6 on CB, here we examine the effect of IL‐6 on [Ca²⁺]_i and catecholamine (CA) secretion in rat CB glomus cells. Calcium imaging showed that extracellular application of IL‐6 induced a rise in [Ca²⁺]_i in cultured glomus cells. Amperometry showed that local application of IL‐6 evoked CA release from glomus cells. Furthermore, the CA secretory response to IL‐6 was blocked by 200 μM Cd²⁺, a well‐known Ca²⁺ channel blocker. Our experiments provide further evidence for the responsiveness of the CB to proinflammatory cytokines and indicate that the CB might play a role in inflammation sensing and transmission of such information to the brain. © 2009 Wiley‐Liss, Inc.