Genistein Excitation of Gonadotrophin‐Releasing Hormone Neurones in Juvenile Female Mice

We investigated the effects of the phytoestrogen genistein on gonadotrophin‐releasing hormone (GnRH) neurones using single‐cell electrophysiology on GnRH‐green fluorescent protein (GFP) transgenic juvenile female mice. Perforated patch‐clamp recordings from GnRH‐GFP neurones showed that approximately 83% of GnRH neurones responded to 30 μm genistein with a markedly prolonged membrane depolarisation. This effect not only persisted in the presence of tetrodotoxin, but also in the presence of amino acid receptor antagonists, indicating the direct site of action on postsynaptic GnRH neurones. Using a voltage clamp technique, we found that 30 μm genistein increased the frequency of synaptic current of GnRH neurones clamped at ?60 mV in the presence of glutamate receptor blocker but not GABA_A receptor blocker. Pre‐incubation of GnRH neurones with 30 μm genistein enhanced kisspeptin‐induced membrane depolarisation and firing. GnRH neurones of juvenile mice injected with genistein in vivo showed an enhanced kisspeptin response compared to vehicle‐injected controls. The transient receptor potential channel (TRPC) blocker 2‐aminoethoxydiphenyl borate (75 μm ) blocked the genistein‐mediated response on GnRH neurones. These results demonstrate that genistein acts on GnRH neurones in juvenile female mice to induce excitation via GABA neurotransmission and TRPCs to enhance kisspeptin‐induced activation.     

Rapid Action of Oestrogen in Luteinising Hormone-Releasing Hormone Neurones: The Role of GPR30

Previously, we have shown that 17β-oestradiol (E₂) induces an increase in firing activity and modifies the pattern of intracellular calcium ([Ca²⁺]_i) oscillations with a latency < 1 min in primate luteinising hormone-releasing hormone (LHRH) neurones. A recent study also indicates that E₂, the nuclear membrane impermeable oestrogen, oestrogen-dendrimer conjugate, and the plasma membrane impermeable oestrogen, E₂-BSA conjugate, all similarly stimulated LHRH release within 10 min of exposure in primate LHRH neurones, indicating that the rapid action of E₂ is caused by membrane signalling. The results from a series of studies further suggest that the rapid action of E₂ in primate LHRH neurones appears to be mediated by GPR30. Although the oestrogen receptor antagonist, ICI 182, 780, neither blocked the E₂-induced LHRH release nor the E₂-induced changes in [Ca²⁺]_i oscillations, E₂ application to cells treated with pertussis toxin failed to result in these changes in primate LHRH neurones. Moreover, knockdown of GPR30 in primate LHRH neurones by transfection with human small interference RNA for GPR30 completely abrogated the E₂-induced changes in [Ca²⁺]_i oscillations, whereas transfection with control siRNA did not. Finally, the GPR30 agonist, G1, resulted in changes in [Ca²⁺]_i oscillations similar to those observed with E₂. In this review, we discuss the possible role of G-protein coupled receptors in the rapid action of oestrogen in neuronal cells.     

Kisspeptin and the Preovulatory Gonadotrophin‐Releasing Hormone/Luteinising Hormone Surge in the Ewe: Basic Aspects and Potential Applications in the Control of Ovulation

The identification of the neural mechanisms controlling ovulation in mammals has long been a ‘holy grail’ over recent decades, although the recent discovery of the kisspeptin systems has totally changed our views on this subject. Kisspeptin cells are the major link between gonadal steroids and gonadotrophin‐releasing hormone (GnRH) neurones. In the female rodent, kisspeptin cells of the preoptic area are involved in the positive‐feedback action of oestrogen on GnRH secretion, although the picture appears more complicated in the ewe. As in rodents, activation of preoptic kisspeptin neurones accompanies the GnRH surge in the ewe but an active role for arcuate kisspeptin neurones has also been proposed. Experimentally, kisspeptin is able to restore reproductive function when the hypothalamic‐hypophyseal ovarian axis is quiescent. For example, i.v. infusion of a low dose of peptide in anoestrous ewes induces an immediate and sustained release of gonadotrophin, which subsides and then provokes a luteinising hormone (LH) surge a few hours later. This pharmacological intervention induces the same hormonal changes normally observed during the follicular phase of the oestrous cycle, including the secretion of oestrogen and its negative‐ and positive‐feedback actions on the secretion of LH and follicle‐stimulating hormone. Accordingly, a high percentage of kisspeptin‐infused animals ovulated. Although the multiple facets of how the kisspeptin systems modulate GnRH secretion are not totally understood, the demonstration that exogenous kisspeptin administration can induce ovulation in anovulatory animals paves the way for future therapeutic applications aiming to control reproduction.     

Roles for Primary Cilia in Gonadotrophin‐Releasing Hormone Neurones in the Mouse

During embryonic development, gonadotrophin‐releasing hormone (GnRH) neurones make an extraordinary migration out of the nose and into the brain where, in adulthood, they drive the pituitary regulation of gonadal function and fertility. Primary cilia are antennae‐like, immotile organelles that project from the surface of nearly all cells, including GnRH neurones. Links between defects in primary cilia and a variety of human pathologies have been discovered that suggest a role for primary cilia in embryogenesis and reproductive function. The present study aimed to investigate whether GnRH neurone primary cilia are critical for their embryonic migration and the adult control of fertility. To achieve this, we used a Cre‐loxP strategy to selectively disrupt primary cilia by deleting Kif3a, an intraflagellar transport protein family member essential for primary cilia assembly and function, specifically in GnRH neurones. Confocal analysis revealed that, in Kif3a^fl/fl (WT‐Kif3a) controls, all GnRH neurones possessed primary cilia, whereas, in GnRH‐Cre^+/?;Kif3a^fl/fl (GnRH‐Kif3aKO) mice, 60% of GnRH neurones lacked any evidence of primary cilia and the remaining 40% possessed only stunted primary cilia (< 2 μm). Despite abolishing normal primary cilia assembly in GnRH neurones from embryogenesis, adult GnRH neurone distribution and reproductive function was remarkably normal. The total number of GnRH neurones was the same in GnRH‐Kif3aKO and WT‐Kif3a controls; however, a significant increase (25%) was identified in the number of GnRH neurones sampled through the midpoint of the rostral pre‐optic area in GnRH‐Kif3aKO mice (P < 0.05). The time to vaginal opening was not different in GnRH‐Kif3aKO mice, although they displayed significantly advanced first oestrus (P < 0.05), and oestrous cycle length was increased (P < 0.05). However, females displayed normal basal levels of luteinising hormone, responded normally to oestrogen‐induced negative‐ and positive‐feedback, and displayed normal fecundity. Taken together, these data suggest that primary cilia and associated signal transduction pathways play a role in the topographical distribution and specific functions of GnRH neurones; however, they are not essential for fertility.     

Male Effect Pheromone Tickles the Gonadotrophin‐Releasing Hormone Pulse Generator

In sheep and goats, the primer pheromone produced by the male induces out‐of‐seasonal ovulation in anoestrous females, the so‐called ‘male effect.’ Because the initial endocrine event following reception of the pheromone is the stimulation of pulsatile luteinising hormone (LH) secretion, the central target of the pheromone is considered to be the putative gonadotrophin‐releasing hormone (GnRH) pulse generator. Using electrophysiological techniques to record multiple‐unit activity (MUA) in close proximity to kisspeptin neurones in the arcuate nucleus (ARC) of Shiba goats, we found that bursts (volleys) of MUA occur at regular intervals, and repetitive bursts are invariably associated with discrete pulses of LH, suggesting that the ARC kisspeptin neurones may be the intrinsic source of the GnRH pulse generator. A brief exposure of female goats to the pheromone immediately elicited an instantaneous rise in MUA, which is followed by an MUA volley and an accompanying LH pulse, indicating that the pheromone signal is transmitted to a subset of the ARC kisspeptin neurones to activate them. Because it has been suggested that the neurokinin B and dynorphin coexpressed in those neurones play critical roles in generating rhythmic bursts, they may be involved in the intracellular pheromone actions that are responsible for inducing the GnRH pulse.     

Evidence that Insulin Signalling in Gonadotrophin‐Releasing Hormone and Kisspeptin Neurones does not Play an Essential Role in Metabolic Regulation of Fertility in Mice

Insulin in the brain plays an important role in regulating reproductive function, as demonstrated via conditional brain‐specific insulin receptor (Insr) deletion (knockout). However, the specific neuronal target cells mediating the central effects of insulin on the reproductive axis remain unidentified. We first investigated whether insulin can act via direct effects on gonadotrophin‐releasing hormone (GnRH) neurones. After clearly detecting Insr mRNA in an immunopurified GnRH cell fraction, we confirmed the presence of insulin receptor protein (InsR) in approximately 82% of GnRH neurones using dual‐label immunohistochemistry. However, we did not observe any insulin‐induced phospho‐Akt (pAkt) or phospho‐extracellular‐signal‐regulated kinase 1/2 in GnRH neurones, and therefore we investigated whether insulin signals via kisspeptin neurones to modulate GnRH release. Using dual‐label immunohistochemistry, InsRs were detected only in approximately 5% of kisspeptin‐immunoreactive cells. Insulin‐induced pAkt was not observed in any kisspeptin‐immunoreactive cells in either the rostral periventricular region of the third ventricle or arcuate nucleus in response to 200 mU of insulin treatment, although a more pharmacological dose (10 U) induced pronounced (> 20%) pAkt–kisspeptin coexpression in both regions. To confirm that insulin signalling via kisspeptin neurones does not critically modulate reproductive function, we generated kisspeptin‐specific InsR knockout (KIRKO) mice and assessed multiple reproductive and metabolic parameters. No significant differences in puberty onset, oestrous cyclicity or reproductive competency were observed in the female or male KIRKO mice compared to their control littermates. However, significantly decreased fasting insulin (P < 0.05) and a nonsignificant trend towards reduced body weight were observed in male KIRKO mice. Thus, InsR signalling in kisspeptin cells is not critical for puberty onset or reproductive competency, although it may have a small metabolic effect in males.     

Electrophysiological Characteristics of Gonadotrophin‐Releasing Hormone 1–3 Neurones: Insights From a Study of Fish Brains

The vertebrate gonadotrophin‐releasing hormone (GnRH) neurones are considered to consist of one group of hypothalamic neuroendocrine and two groups of extrahypothalamic neuromodulatory GnRH neurones, and each group of neurones expresses different molecular species of GnRH peptide. Different GnRH peptides are produced by one of the three paralogous GnRH genes, gnrh1, gnrh2 and gnrh3, which are considered to have originated from gene duplications. All three GnRH systems are well developed in teleost brains. By taking advantage of this, and especially the use of GnRH‐green fluoresecent protein transgenic fish, the anatomical and electrophysiological properties of all three types of GnRH neurones can now be studied. The hypophysiotropic GnRH1 neurones in the preoptic area show episodic spontaneous electrical activities, whereas the extrahypothalamic GnRH2 neurones in the midbrain and GnRH3 neurones in the terminal nerve show regular intrinsic pacemaker activities. It is suggested that these different electrophysiological properties are related to their different functions (i.e. GnRH1 neurones act as hypophysiotropic neuroendocrine regulators and GnRH2 and GnRH3 neurones act as neuromodulators). The present review focuses on recent electrophysiological analyses of GnRH3 neurones, which have revealed the excitatory GABAergic and the inhibitory FMRFamide‐like peptidergic regulations acting upon them, as well as gap junctional electrotonic coupling.     

From Nose to Brain: Development of Gonadotrophin‐Releasing Hormone ‐1 Neurones

Gonadotrophin‐releasing hormone‐1 (GnRH‐1) is essential for mammalian reproduction, controlling release of gonadotrophins from the anterior pituitary. GnRH‐1 neurones migrate from the nasal placode into the forebrain during development. Although first located within the nasal placode, the embryonic origin/lineage of GnRH‐1 neurones is still unclear. The migration of GnRH‐1 cells is the best characterised example of neurophilic/axophilic migration, with the cells using a subset of olfactory‐derived vomeronasal axons as their pathway and numerous molecules to guide their movement into the forebrain. Exciting work in this area is beginning to identify intersecting pathways that orchestrate the movement of these critical neuroendocrine cells into the central nervous system, both spatially and temporally, through a diverse and changing terrain. Once within the forebrain, little is known about how the axons target the median eminence and ultimately secrete GnRH‐1 in a pulsatile fashion.     

Gonadotrophin‐Releasing Hormone (GnRH) Exerts Stimulatory Effects on GnRH Neurones in Intact Adult Male and Female Mice

There is substantial evidence for a role of the neuropeptide gonadotrophin‐releasing hormone (GnRH) in the regulation of GnRH neurone secretion but how this is achieved is not understood. We examined here the effects of GnRH on the electrical excitability and intracellular calcium concentration ([Ca²⁺]_i) of GnRH neurones in intact adult male and female mice. Perforated‐patch electrophysiological recordings from GnRH‐green fluorescent protein‐tagged GnRH neurones revealed that 3 nm –3 μm GnRH evoked gradual approximately 3 mV depolarisations in membrane potential from up to 50% of GnRH neurones in male and female mice. The depolarising effect of GnRH was observed on approximately 50% of GnRH neurones throughout the oestrous cycle. However, at pro‐oestrus alone, GnRH was also found to transiently hyperpolarise approximately 30% of GnRH neurones. Both hyperpolarising and depolarising responses were maintained in the presence of tetrodotoxin. Calcium imaging studies undertaken in transgenic GnRH‐pericam mice showed that GnRH suppressed [Ca²⁺]_i in approximately 50% of GnRH neurones in dioestrous and oestrous mice. At pro‐oestrus, 25% of GnRH neurones exhibited a suppressive [Ca²⁺]_i response to GnRH, whereas 17% were stimulated. These results demonstrate that nm to μm concentrations of GnRH exert depolarising actions on approximately 50% of GnRH neurones in males and females throughout the oestrous cycle. This is associated with a reduction in [Ca²⁺]_i. At pro‐oestrus, however, a further population of GnRH neurones exhibit a hyperpolarising response to GnRH. Taken together, these studies indicate that GnRH acts predominantly as a neuromodulator at the level of the GnRH cell bodies to exert a predominant excitatory influence upon GnRH neurones in intact adult male and female mice.     

A Dynamical Model for the Control of the Gonadotrophin‐Releasing Hormone Neurosecretory System

The gonadotrophin‐releasing hormone (GnRH) neurosecretory system involves both endocrine neurones and associated brain cells responsible for the control of GnRH release into the pituitary portal blood. Alternation between a pulsatile regime and the pre‐ovulatory surge is the hallmark of GnRH secretion in ovarian cycles of female mammals. In previous studies, we have introduced a mathematical model of the pulse and surge GnRH generator and derived appropriate dynamics‐based constraints on the model parameters, both to reproduce the right sequence of secretion events and to fulfil quantitative specifications on GnRH release. In the present study, we explain how these constraints amount to embedding time‐ and dose‐dependent steroid control within the model. We further examine under which conditions the oestradiol‐driven surge may be withdrawn by pre‐surge progesterone administration and simulate both oestradiol and progesterone challenges in the pulsatile regime.     

Burst Firing in Gonadotrophin‐Releasing Hormone Neurones does not Require Ionotrophic GABA or Glutamate Receptor Activation

Burst firing is a feature of many neuroendocrine cell types, including the hypothalamic gonadotrophin‐releasing hormone (GnRH) neurones that control fertility. The role of intrinsic and extrinsic influences in generating GnRH neurone burst firing is presently unclear. In the present study, we investigated the role of fast amino acid transmission in burst firing by examining the effects of receptor antagonists on bursting displayed by green fluorescent protein GnRH neurones in sagittal brain slices prepared from adult male mice. Blockade of AMPA and NMDA glutamate receptors with a cocktail of CNQX and AP5 was found to have no effects on burst firing in GnRH neurones. The frequency of bursts, dynamics of individual bursts, or percentage of firing clustered in bursts was not altered. Similarly, GABA_A receptor antagonists bicuculline and picrotoxin had no effects upon burst firing in GnRH neurones. To examine the importance of both glutamate and GABA ionotrophic signalling, a cocktail including picrotoxin, CNQX and AP5 was used but, again, this was found to have no effects on GnRH neurone burst firing. To further question the impact of endogenous amino acid release on burst firing, electrical activation of anteroventral periventricular nuclei GABA/glutamate inputs to GnRH neurones was undertaken and found to have no impact on burst firing. Taken together, these observations indicate that bursting in GnRH neurones is not dependent upon acute ionotrophic GABA and glutamate signalling and suggest that extrinsic inputs to GnRH neurones acting through AMPA, NMDA and GABA_A receptors are unlikely to be required for burst initiation in these cells.     

Evidence for Suprachiasmatic Vasopressin Neurones Innervating Kisspeptin Neurones in the Rostral Periventricular Area of the Mouse Brain: Regulation by Oestrogen

In rodents, a circadian signal from the suprachiasmatic nucleus (SCN) is essential for the pro‐oestrous surge of gonadotrophin‐releasing hormone (GnRH), which, in turn, induces luteinising hormone (LH) surge and ovulation. We hypothesised that kisspeptin (KP) neurones in the anteroventral periventricular and periventricular preoptic nuclei (AVPV/PeN) form part of the communication pathway between the SCN and GnRH neurones. In anterograde track tracing studies, we first identified vasopressin (VP)‐containing axons of SCN origin in apposition to KP‐immunoreactive (IR) neurones. Studies to quantify this input relied on the observation that VP‐synthesising neurones in the SCN differ from other VP systems in their lack of galanin expression. In ovariectomised mice, 30.79 ± 1.63% of KP‐IR perikarya and proximal dendrites within the AVPV/PeN received galanin‐negative VP‐IR varicosities. Oestrogen‐treatment significantly increased the number of KP‐IR neurones, with their percentage apposed by galanin‐negative VP‐IR varicosities (46.95 ± 1.88%) and the number of VP‐IR appositions on individual KP‐IR neurones. At the ultrastructural level, the VP‐IR terminals formed symmetric synapses with KP‐IR neurones, which was in accordance with the morphology of inhibitory synapses established by SCN neurones. By contrast to VP, vasoactive intestinal polypeptide (VIP), which is synthesised by a distinct subset of SCN neurones, occurred only rarely in axons apposed to KP‐IR neurones. Altogether, our results are consistent with the hypothesis that KP neurones located in the mouse AVPV/PeN receive circadian information from the SCN via a vasopressinergic monosynaptic pathway, which is enhanced by oestrogen.     

Kisspeptin/Metastin: A Key Molecule Controlling Two Modes of Gonadotrophin-Releasing Hormone/Luteinising Hormone Release in Female Rats

Kisspeptin (also known as metastin), a hypothalamic peptide, has attracted attention as a key molecule in the release of gonadotrophin-releasing hormone (GnRH) in various mammalian species, such as rodents, sheep and primates. Two populations of kisspeptin neurones in the brain may control two modes of GnRH release to time the onset of puberty and regulate oestrous cyclicity in rats and mice. One population of kisspeptin neurones, located in the anteroventral periventricular nucleus, appears to be responsible for the induction of the GnRH surge that leads to the luteinising hormone surge and ovulation. The other, located in the hypothalamic arcuate nucleus, appears to be involved in generating GnRH pulses, resulting in luteinising hormone pulses followed by follicular development and steroidogenesis in the ovary. The present review focuses on the physiological role of the two populations of kisspeptin neurones in controlling gonadal functions by generating the two modes of GnRH release in a female rat model.     

Mutational Analysis of the Genes Encoding RFAmide‐Related Peptide‐3, the Human Orthologue of Gonadotrophin‐Inhibitory Hormone,and its Receptor (GPR147) in Patients with Gonadotrophin‐Releasing Hormone‐Dependent Pubertal Disorders

RFamide‐related peptide‐3 (RFRP‐3), the orthologue of avian gonadotrophin‐inhibitory hormone, and its receptor GPR147 have been recently identified in the human hypothalamus, and their roles in the regulation of reproductive axis has been studied. The present study aimed to investigate whether the presence of variants in the genes encoding human RFRP‐3 (NPVF gene) and its receptor, GPR147 (NPFFR1 gene), is associated with the occurrence of gonadotrophin‐releasing hormone‐dependent pubertal disorders. Seventy‐eight patients with idiopathic central precocious puberty (CPP) and 51 with normosmic isolated hypogonadotrophic hypogonadism (nIHH) were investigated. Fifty healthy subjects comprised the control group. The coding sequences of the NPVF and NPFFR1 genes were amplified and sequenced. Odds ratios (OR) were used to estimate the likelihood of CPP or nIHH in the presence of the described polymorphisms. All such polymorphisms have already been registered in the National Center for Biotechnology Information database. A three‐nucleotide in frame deletion was identified in the NPVF gene (p.I71_K72), with a smaller proportion in the CPP (5%) compared to the nIHH (15%) group (P = 0.06). This results in the deletion of the isoleucine at position 71, adjacent to lysine at an endoproteolytic cleavage site of the precursor peptide. This polymorphism was associated with a lower risk of CPP (OR = 0.33; 95% confidence interval = 0.08–0.88); interestingly, only two men with nIHH were homozygotes for this variant. A total of five missense polymorphisms were found in the NPFFR1 gene, which encodes GPR147, with similar frequencies among groups and no association with pubertal timing. Our data suggest that RFRP‐3/GPR147 may play secondary, modulatory roles on the regulation of pubertal development; a restraining modulatory effect of the NPVF p.I71_K72 variant on the activation of the gonadotrophic axis cannot be ruled out and deserves further investigation.     

Hypothalamic KISS1 Expression,Gonadotrophin‐Releasing Hormone and Neurotransmitter Innervation Vary with Stress and Sensitivity in Macaques

The present study examined the effect of short‐term psychosocial and metabolic stress in a monkey model of stress‐induced amenorrhaea on the hypothalamic‐pituitary‐gonadal axis. KISS1 expression was determined by in situ hybridisation in the infundibular arcuate nucleus. Downstream of KISS1, gonadotrophin‐releasing hormone (GnRH) axons in lateral areas rostral to the infundibular recess, serum luteinising hormone (LH) and serum oestradiol were measured by immunohistochemistry and radioimmunoassay. Upstream of KISS1, norepinephrine axons in the rostral arcuate nucleus and serotonin axons in the anterior hypothalamus and periaqueductal grey were measured by immunohistochemistry. Female cynomolgus macaques (Macaca fascicularis) characterised as highly stress resilient (HSR) or stress sensitive (SS) were examined. After characterisation of stress sensitivity, monkeys were either not stressed, or mildly stressed for 5 days before euthanasia in the early follicular phase. Stress consisted of 5 days of 20% food reduction in a novel room with unfamiliar conspecifics. There was a significant increase in KISS1 expression in HSR and SS animals in the presence versus absence of stress (P = 0.005). GnRH axon density increased with stress in HSR and SS animals (P = 0.015), whereas LH showed a gradual but nonsignificant increase with stress. Oestradiol trended higher in HSR animals and there was no effect of stress (P = 0.83). Norepinephrine axon density (marked with dopamine β‐hydroxylase) increased with stress in both HSR and SS groups (P ≤ 0.002), whereas serotonin axon density was higher in HSR compared to SS animals and there was no effect of stress (P = 0.03). The ratio of dopamine β‐hydroxylase/oestradiol correlated with KISS1 (P = 0.052) and GnRH correlated with serum LH (P = 0.039). In conclusion, oestradiol inhibited KISS1 in the absence of stress, although stress increased norepinephrine, which may over‐ride oestradiol inhibition of KISS1 expression. We speculate that neural pathways transduce stress to KISS1 neurones, which changes their sensitivity to oestradiol.     

The Distribution of Kisspeptin (Kiss)1‐ and Kiss2‐Positive Neurones and Their Connections with Gonadotrophin‐Releasing Hormone‐3 Neurones in the Zebrafish Brain

Kisspeptin is a neuroendocrine hormone with a critical role in the activation of gonadotrophin‐releasing hormone (GnRH) neurones, which is vital for the onset of puberty in mammals. However, the functions of kisspeptin neurones in non‐mammalian vertebrates are not well understood. We have used transgenics to labell kisspeptin neurones (Kiss1 and Kiss2) with mCherry in zebrafish (Danio rerio). In kiss1:mCherry transgenic zebrafish, Kiss1 cells were located in the dorsomedial and ventromedial habenula, with their nerve fibres contributing to the fasciculus retroflexus and projecting to the ventral parts of the interpeduncular and raphe nuclei. In kiss2:mCherry zebrafish, Kiss2 cells were primarily located in the dorsal zone of the periventricular hypothalamus and, to a lesser extent, in the periventricular nucleus of the posterior tuberculum and the preoptic area. Kiss2 fibres formed a wide network projecting into the telencephalon, the mesencephalon, the hypothalamus and the pituitary. To study the relationship of kisspeptin neurones and GnRH3 neurones, these fish were crossed with gnrh3:EGFP zebrafish to obtain kiss1:mCherry/gnrh3:EGFP and kiss2:mCherry/gnrh3:EGFP double transgenic zebrafish. The GnRH3 fibres ascending to the habenula were closely associated with Kiss1 fibres projecting from the ventral habenula. On the other hand, GnRH3 fibres and Kiss2 fibres were adjacent but scarcely in contact with each other in the telencephalon and the hypothalamus. The Kiss2 and GnRH3 fibres in the ventral hypothalamus projected into the pituitary via the pituitary stalk. In the pituitary, Kiss2 fibres were directly in contact with GnRH3 fibres in the pars distalis. These results reveal the pattern of kisspeptin neurones and their connections with GnRH3 neurones in the brain, suggesting distinct mechanisms for Kiss1 and Kiss2 in regulating reproductive events in zebrafish.     

Compound Deficiencies in Multiple Fibroblast Growth Factor Signalling Components Differentially Impact the Murine Gonadotrophin‐Releasing Hormone System

Gonadotrophin‐releasing hormone (GnRH) neurones control the onset and maintenance of fertility. Aberrant development of the GnRH system underlies infertility in Kallmann syndrome [KS; idiopathic hypogonadotropic hypogonadism (IHH) and anosmia]. Some KS patients harbour mutations in the fibroblast growth factor receptor 1 (Fgfr1) and Fgf8 genes. The biological significance of these two genes in GnRH neuronal development was corroborated by the observation that GnRH neurones were severely reduced in newborn transgenic mice deficient in either gene. In the present study, we hypothesised that the compound deficiency of Fgf8 and its cognate receptors, Fgfr1 and Fgfr3, may lead to more deleterious effects on the GnRH system, thereby resulting in a more severe reproductive phenotype in patients harbouring these mutations. This hypothesis was tested by counting the number of GnRH neurones in adult transgenic mice with digenic heterozygous mutations in Fgfr1/Fgf8, Fgfr3/Fgf8 or Fgfr1/Fgfr3. Monogenic heterozygous mutations in Fgfr1, Fgf8 or Fgfr3 caused a 30–50% decrease in the total number of GnRH neurones. Interestingly, mice with digenic mutations in Fgfr1/Fgf8 showed a greater decrease in GnRH neurones compared to mice with a heterozygous defect in the Fgfr1 or Fgf8 alone. This compounding effect was not detected in mice with digenic heterozygous mutations in Fgfr3/Fgf8 or Fgfr1/Fgfr3. These results support the hypothesis that IHH/KS patients with digenic mutations in Fgfr1/Fgf8 may have a further reduction in the GnRH neuronal population compared to patients harbouring monogenic haploid mutations in Fgfr1 or Fgf8. Because only Fgfr1/Fgf8 compound deficiency leads to greater GnRH system defect, this also suggests that these fibroblast growth factor signalling components interact in a highly specific fashion to support GnRH neuronal development.     

Seasonal Plasticity in the Peptide Neuronal Systems: Potential Roles of Gonadotrophin‐Releasing Hormone,Gonadotrophin‐Inhibiting Hormone,Neuropeptide Y and Vasoactive Intestinal Peptide in the Regulation of the Reproductive Axis in Subtropical Indian Weaver Birds

Two experiments examined the expression of gonadotrophin‐releasing and inhibiting hormones (GnRH‐I, GnRH‐II and GnIH), neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) in subtropical Indian weaver birds, which demonstrate relative photorefractoriness. Experiment 1 measured peptide expression levels in the form of immunoreactive (‐IR) cells, percentage cell area and cell optical density in the preoptic area (GnRH‐I), midbrain (GnRH‐II), paraventricular nucleus (GnIH), mediobasal hypothalamus [dorsomedial hypothalamus (DMH), infundibular complex (INc), NPY and VIP] and lateral septal organ (VIP) during the progressive, breeding, regressive and nonbreeding phases of the annual reproductive cycle. GnRH‐I was decreased in the nonbreeding and VIP was increased in INc in the breeding and regressive states. GnRH‐II and NPY levels did not differ between the testicular phases. Double‐labelled immunohistochemistry (IHC) revealed a close association between the GnRH/GnIH, GnRH/NPY, GnRH/VIP and GnIH/NPY peptide systems, implicating them interacting and playing roles in the reproductive regulation in weaver birds. Experiment 2 further measured these peptide levels in the middle of day and night in weaver birds that were maintained under short days (8 : 16 h light /dark cycle; photosensitive), exposed to ten long days (16 : 8 h light /dark cycle; photostimulated) or maintained for approximately 2 years on a 16 : 8 h light /dark cycle (photorefractory). Reproductively immature testes in these groups precluded the possible effect of an enhanced gonadal feedback on the hypothalamic peptide expression. There were group differences in the GnRH‐I (not GnRH‐II), GnIH, NPY and VIP immunoreactivity, albeit with variations in immunoreactivity measures in the present study. These results, which are consistent with those reported in birds with relative photorefractoriness, show the distribution and possibly a complex interaction of key neuropeptides in the regulation of the annual reproductive cycle in Indian weaver birds.