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.     

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.     

Recent Discoveries on the Control of Gonadotrophin‐Releasing Hormone Neurones in Nonhuman Primates

Since Ernst Knobil proposed the concept of the gonadotrophin‐releasing hormone (GnRH) pulse‐generator in the monkey hypothalamus three decades ago, we have made significant progress in this research area with cellular and molecular approaches. First, an increase in pulsatile GnRH release triggers the onset of puberty. However, the question of what triggers the pubertal increase in GnRH is still unclear. GnRH neurones are already mature before puberty but GnRH release is suppressed by a tonic GABA inhibition. Our recent work indicates that blocking endogenous GABA inhibition with the GABA_A receptor blocker, bicuculline, dramatically increases kisspeptin release, which plays an important role in the pubertal increase in GnRH release. Thus, an interplay between the GABA, kisspeptin, and GnRH neuronal systems appears to trigger puberty. Second, cultured GnRH neurones derived from the olfactory placode of monkey embryos exhibit synchronised intracellular calcium, [Ca²⁺]_i, oscillations and release GnRH in pulses at approximately 60‐min intervals after 14 days in vitro (div). During the first 14 div, GnRH neurones undergo maturational changes from no [Ca²⁺]_i oscillations and little GnRH release to the fully functional state. Recent work also shows GnRH mRNA expression increases during in vitro maturation. This mRNA increase coincides with significant demethylation of a CpG island in the GnRH 5′‐promoter region. This suggests that epigenetic differentiation occurs during GnRH neuronal maturation. Third, oestradiol causes rapid, direct, excitatory action in GnRH neurones and this action of oestradiol appears to be mediated through a membrane receptor, such as G‐protein coupled receptor 30.     

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.     

Foetal Hypothalamic and Pituitary Expression of Gonadotrophin‐Releasing Hormone and Galanin Systems is Disturbed by Exposure to Sewage Sludge Chemicals via Maternal Ingestion

Animals and humans are chronically exposed to endocrine disrupting chemicals (EDCs) that are ubiquitous in the environment. There are strong circumstantial links between environmental EDC exposure and both declining human/wildlife reproductive health and the increasing incidence of reproductive system abnormalities. The verification of such links, however, is difficult and requires animal models exposed to ‘real life’, environmentally relevant concentrations/mixtures of environmental contaminants (ECs), particularly in utero, when sensitivity to EC exposure is high. The present study aimed to determine whether the foetal sheep reproductive neuroendocrine axis, particularly gondotrophin‐releasing hormone (GnRH) and galaninergic systems, were affected by maternal exposure to a complex mixture of chemicals, applied to pasture, in the form of sewage sludge. Sewage sludge contains high concentrations of a spectrum of EDCs and other pollutants, relative to environmental concentrations, but is frequently recycled to land as a fertiliser. We found that foetuses exposed to the EDC mixture in utero through their mothers had lower GnRH mRNA expression in the hypothalamus and lower GnRH receptor (GnRHR) and galanin receptor (GALR) mRNA expression in the hypothalamus and pituitary gland. Strikingly, this, treatment had no significant effect on maternal GnRH or GnRHR mRNA expression, although GALR mRNA expression within the maternal hypothalamus and pituitary gland was reduced. The present study clearly demonstrates that the developing foetal neuroendocrine axis is sensitive to real‐world mixtures of environmental chemicals. Given the important role of GnRH and GnRHR in the regulation of reproductive function, its known role programming role in utero, and the role of galanin in the regulation of many physiological/neuroendocrine systems, in utero changes in the activity of these systems are likely to have long‐term consequences in adulthood and represent a novel pathway through which EC mixtures could perturb normal reproductive function.     

Neuronal Gonadotrophin‐Releasing Hormone (GnRH) and Astrocytic Gonadotrophin Inhibitory Hormone (GnIH) Immunoreactivity in the Adult Rat Hippocampus

Gonadotrophin‐releasing hormone (GnRH) and gonadotrophin inhibitory hormone (GnIH) are neuropeptides secreted by the hypothalamus that regulate reproduction. GnRH receptors are not only present in the anterior pituitary, but also are abundantly expressed in the hippocampus of rats, suggesting that GnRH regulates hippocampal function. GnIH inhibits pituitary gonadotrophin secretion and is also expressed in the hippocampus of a songbird; its role outside of the reproductive axis is not well established. In the present study, we employed immunohistochemistry to examine three forms of GnRH [mammalian GnRH‐I (mGnRH‐I), chicken GnRH‐II (cGnRH‐II) and lamprey GnRH‐III (lGnRH‐III)] and GnIH in the adult rat hippocampus. No mGnRH‐I and cGnRH‐II+ cell bodies were present in the hippocampus. Sparse mGnRH‐I and cGnRH‐II+ fibres were present within the CA1 and CA3 fields of the hippocampus, along the hippocampal fissure, and within the hilus of the dentate gyrus. No lGnRH‐III was present in the rodent hippocampus. GnIH‐immunoreactivity was present in the hippocampus in cell bodies that resembled astrocytes. Males had more GnIH+ cells in the hilus of the dentate gyrus than females. To confirm the GnIH+ cell body phenotype, we performed double‐label immunofluorescence against GnIH, glial fibrillary acidic protein (GFAP) and NeuN. Immunofluorescence revealed that all GnIH+ cell bodies in the hippocampus also contained GFAP, a marker of astrocytes. Taken together, these data suggest that GnRH does not reach GnRH receptors in the rat hippocampus primarily via synaptic release. By contrast, GnIH might be synthesised locally in the rat hippocampus by astrocytes. These data shed light on the sites of action and possible functions of GnRH and GnIH outside of the hypothalamic‐pituitary‐gonadal axis.     

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.     

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.     

Kisspeptin/Gpr54‐Independent Gonadotrophin‐Releasing Hormone Activity in Kiss1 and Gpr54 Mutant Mice

The kisspeptin/Gpr54 signalling pathway plays a critical role in reproduction by stimulating the secretion of gonadotrophin‐releasing hormone (GnRH), yet mice carrying mutations in Kiss1 (which encodes kisspeptin) or Gpr54 exhibit partial sexual maturation. For example, a proportion of female Kiss1^?/? and Gpr54^?/? mice exhibit vaginal oestrus, and some male Kiss1^?/? and Gpr54^?/? mice exhibit spermatogenesis. To characterise this partial sexual maturation, we examined the vaginal cytology of female Kiss1^?/? and Gpr54^?/? mice over time. Almost all mutant mice eventually enter oestrus, and then spontaneously transition from oestrus to dioestrus and back to oestrus again. These transitions are not associated with ovulation, and the frequency of these transitions increases with age. The oestrus exhibited by female Kiss1^?/? and Gpr54^?/? mice was disrupted by the administration of the competitive GnRH antagonist acyline, which also resulted in lower uterine weights and, in Kiss1^?/? mice, lower serum follicle‐stimulating hormone (FSH) and luteinising hormone (LH) concentrations. Similarly, male Kiss1^?/? and Gpr54^?/? mice treated with acyline had smaller testicular sizes and an absence of mature sperm. In addition to examining intact Kiss1^?/? and Gpr54^?/? mice, we also assessed the effects of acyline on gonadotrophin concentrations in gonadectomised mice. Gonadectomy resulted in a significant increase in serum FSH concentrations in male Gpr54^?/? and Kiss1^?/? mice. Acyline administration to gonadectomised Kiss1^?/? and Gpr54^?/? male mice lowered serum FSH and LH concentrations significantly. By contrast to males, gonadectomy did not result in significant gonadotrophin changes in female Kiss1^?/? and Gpr54^?/? mice, but acyline administration was followed by a decrease in LH concentrations. These results demonstrate that, although kisspeptin signalling is critical for the high levels of GnRH activity required for normal sexual maturation and for ovulation, Kiss1^?/? and Gpr54^?/? mice retain some degree of GnRH activity. This GnRH activity is sufficient to produce significant effects on vaginal cytology and uterine weights in female mice and on spermatogenesis and testicular weights in male mice.     

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.     

Gonadotrophin‐Releasing Hormone Neurones in a Photoperiodic Songbird Express Fos and Egr‐1 Protein After a Single Long Day

Birds use a variety of environmental cues, such as day length, temperature and social interactions, to time reproductive efforts. For most seasonally breeding birds, day length is the most important cue and takes precedence over all others. Experimental manipulation of day length has shown that, in a number of galliformes and passeriformes, exposure to a single long day induces a rise in plasma luteinising hormone (LH). The mechanisms underlying this response are only beginning to be understood. In Japanese quail and Zonotrichia sparrows, one long day causes striking up‐regulation of the protein products of immediate early genes (IEGs) in the mediobasal hypothalamus, near gonadotrophin‐releasing hormone (GnRH) axons and terminals. Photoperiodic induction of the same proteins in the GnRH somata themselves, however, has not been described in these species. In the present study, we used immunohistochemistry to assay the induction of two IEGs, Fos and Egr‐1, in the GnRH somata of male and female white‐throated sparrows (Zonotrichia albicollis) exposed to a single long day. We found that immunoreactivity for both proteins increased in a subset of the GnRH neurones of the septo‐preoptic area by the morning after the long day. Photo‐induced expression of Egr‐1 or Fos protein in GnRH neurones was limited to a population of cells in the medial preoptic area. Males showed significantly greater induction of both proteins in this population of GnRH neurones than did females, which is consistent with the hypothesis that males may be more sensitive to photic cues. Overall, the results obtained suggest that photostimulation stimulates new protein synthesis in GnRH neurones on a relatively rapid time scale. Further research is required to determine whether the GnRH somata are themselves integrating photic cues, or whether they are responding rapidly to an increased demand for GnRH synthesis.     

Unaltered Hypothalamic Metabolic Gene Expression in Kiss1r Knockout Mice Despite Obesity and Reduced Energy Expenditure

Kisspeptin controls reproduction by stimulating gonadotrophin‐releasing hormone neurones via its receptor Kiss1r. Kiss1r is also expressed other brain areas and in peripheral tissues, suggesting additional nonreproductive roles. We recently determined that Kiss1r knockout (KO) mice develop an obese and diabetic phenotype. In the present study, we investigated whether Kiss1r KOs develop this metabolic phenotype as a result of alterations in the expression of metabolic genes involved in the appetite regulating system of the hypothalamus, including neuropeptide Y (Npy) and pro‐opiomelanocortin (Pomc), as well as leptin receptor (Lepr), ghrelin receptor (Ghsr), and melanocortin receptors 3 and 4 (Mc3r, Mc4r). Body weights, leptin levels and hypothalamic gene expression were measured in both gonad‐intact and gonadectomised (GNX) mice at 8 and 20 weeks of age that had received either normal chow or a high‐fat diet. We detected significant increases in Pomc expression in gonad‐intact Kiss1r KO mice at 8 and 20 weeks, although there were no alterations in the other metabolic‐related genes. However, the Pomc increases appeared to reflect genotype differences in circulating sex steroids, because GNX wild‐type and Kiss1r KO mice exhibited similar Pomc levels, along with similar Npy levels. The altered Pomc gene expression in gonad‐intact Kiss1r KO mice is consistent with previous reports of reduced food intake in these mice and may serve to increase the anorexigenic drive, perhaps compensating for the obese state. However, the surprising overall lack of changes in any of the hypothalamic metabolic genes in GNX KO mice suggests that the aetiology of obesity in the absence of kisspeptin signalling may reflect peripheral rather than central metabolic impairments.     

Daily Patterns and Adaptation of the Ghrelin,Growth Hormone and Insulin‐Like Growth Factor‐1 System Under Daytime Food Synchronisation in Rats

Daytime restricted feeding promotes the re‐alignment of the food entrained oscillator (FEO). Endocrine cues which secretion is regulated by the transition of fasting and feeding cycles converge in the FEO. The present study aimed to investigate the ghrelin, growth hormone (GH) and insulin‐like growth factor (IGF)‐1 system because their release depends on rhythmic and nutritional factors, and the output from the system influences feeding and biochemical status. In a daily sampling approach, rats that were fed ad lib. were compared with rats on a reversed (daytime) and restricted feeding schedule by 3 weeks (dRF; food access for 2 h), also assessing the effect of acute fasting and refeeding. We undertook measurements of clock protein BMAL1 and performed somatometry of peripheral organs and determined the concentration of total, acylated and unacylated ghrelin, GH and IGF‐1 in both serum and in its main synthesising organs. During dRF, BMAL1 expression was synchronised to mealtime in hypophysis and liver; rats exhibited acute hyperphagia, stomach distension with a slow emptying, a phase shift in liver mass towards the dark period and decrease in mass perigonadal white adipose tissue. Total ghrelin secretion during the 24‐h period increased in the dRF group as a result of elevation of the unacylated form. By contrast, GH and IGF‐1 serum concentration fell, with a modification of GH daily pattern after mealtime. In the dRF group, ghrelin content in the stomach and pituitary GH content decreased, whereas hepatic IGF‐1 remained equal. The daily patterns and synthesis of these hormones had a rheostatic adaptation. The endocrine adaptive response elicited suggests that it may be associated with the regulation of metabolic, behavioural and physiological processes during the paradigm of daytime restricted feeding and associated FEO activity.     

Chronic Exposure to Low Levels of Oestradiol-17β Affects Oestrous Cyclicity, Hypothalamic Norepinephrine and Serum Luteinising Hormone in Young Intact Rats

Chronic exposure to oestrogens is known to inhibit the secretion of luteinising hormone (LH) in rats, leading to anovulation. Hypothalamic catecholamines, norepinephrine and dopamine play an important role in LH regulation. However, the effects of chronic exposure to low levels of oestradiol on hypothalamic catecholamines have not been investigated thoroughly. In the present study, adult female Sprague–Dawley rats were either sham implanted or implanted with 17β‐oestradiol (E₂) pellets (20 ng/day) for 30 (E‐30), 60 (E‐60) or 90 (E‐90) days. E₂ exposure affected oestrous cyclicity and ovarian morphology in a duration‐dependent manner. There was no change in oestrous cyclicity in E‐30 rats; however, 75% of E‐60 and 95% of E‐90 rats were acyclic (P < 0.05). Cycling rats from E‐30 or the control group were killed at different time points on the afternoon of pro‐oestrous. E‐30 rats in oestrous, constant oestrous rats in the E‐60 and E‐90 groups and a group of old constant oestrous (OCE) rats were killed at 12.00 h. LH was measured in the serum by radioimmunoassay. Individual hypothalamic nuclei that are involved in LH regulation were microdissected and analysed for norepinephrine and dopamine levels using high‐performance liquid chromatography/electrochemical detection. Norepinephrine levels in the hypothalamic nuclei increased significantly in control and E‐30 groups during the afternoon of pro‐oestrous, which was accompanied by a rise in LH levels (P < 0.05). On the day of oestrous, norepinephrine concentrations in hypothalamic nuclei and serum LH were significantly lower in E‐60, E‐90 and OCE rats compared to E‐30 and control rats. On the other hand, dopamine levels declined significantly in one hypothalamic nucleus. These results indicate that chronic E₂ exposure affects hypothalamic catecholamine and serum LH levels in a duration‐dependent manner. This coincides well with the loss of cyclicity observed in these animals. These results suggest that repeated exposure to endogenous oestrogens could play a role in reproductive senescence.     

Lentivirus‐Mediated α‐Melanocyte‐Stimulating Hormone Overexpression in the Hypothalamus Decreases Diet Induced Obesity in Mice

Melanocyte stimulating hormone (MSH) derived from the pro‐hormone pro‐opiomelanocortin (POMC) has potent effects on metabolism and feeding that lead to reduced body weight in the long‐term. To determine the individual roles of POMC derived peptides and their sites of action, we created a method for the delivery of single MSH peptides using lentiviral vectors and studied the long‐term anti‐obesity effects of hypothalamic α‐MSH overexpression in mice. An α‐MSH lentivirus (LVi‐α‐MSH‐EGFP) vector carrying the N‘‐terminal part of POMC and the α‐MSH sequence was generated and shown to produce bioactive peptide in an in vitro melanin synthesis assay. Stereotaxis was used to deliver the LVi‐α‐MSH‐EGFP or control LVi‐EGFP vector to the arcuate nucleus (ARC) of the hypothalamus of male C57Bl/6N mice fed on a high‐fat diet. The effects of 6‐week‐treatment on body weight, food intake, glucose tolerance and organ weights were determined. Additionally, a 14‐day pairfeeding study was conducted to assess whether the weight decreasing effect of the LVi‐α‐MSH‐EGFP treatment is dependent on decreased food intake. The 6‐week LVi‐α‐MSH‐EGFP treatment reduced weight gain (8.4 ± 0.4 g versus 12.3 ± 0.6 g; P < 0.05), which was statistically significant starting from 1 week after the injections. The weight of mesenteric fat was decreased and glucose tolerance was improved compared to LVi‐EGFP treated mice. Food intake was decreased during the first week in the LVi‐α‐MSH‐EGFP treated mice but subsequently increased to the level of LVi‐EGFP treated mice. The LVi‐EGFP injected control mice gained more weight even when pairfed to the level of food intake by LVi‐α‐MSH‐EGFP treated mice. We demonstrate that gene transfer of α‐MSH, a single peptide product of POMC, into the ARC of the hypothalamus, reduces obesity and improves glucose tolerance, and that factors other than decreased food intake also influence the weight decreasing effects of α‐MSH overexpression in the ARC. Furthermore, viral MSH vectors delivered stereotaxically provide a novel tool for further exploration of chronic site‐specific effects of POMC peptides.     

Gonadotrophin‐Releasing Hormone Nerve Terminals,Tanycytes and Neurohaemal Junction Remodelling in the Adult Median Eminence: Functional Consequences for Reproduction and Dynamic Role of Vascular Endothelial Cells

Although coordinated actions of several areas within the hypothalamus are involved in the secretion of gonadotrophin‐releasing hormone (GnRH), the median eminence of the hypothalamus, where the nerve terminals are located, plays a particularly critical role in the release of GnRH. In adult females, prior to the preovulatory surge of GnRH, the retraction of specialised ependymoglial cells lining the floor of the third ventricle named tanycytes allows for the juxtaposition of GnRH nerve terminals with the adjacent pericapillary space of the pituitary portal vasculature, thus forming direct neurohaemal junctions. These morphological changes occur within a few hours and are reversible. Such remodelling may promote physiological conditions to enhance the central release of GnRH and potentiate oestrogen‐activated GnRH release. This plasticity involves dynamic cell interactions that bring into play tanycytes, astrocytes, vascular endothelial cells and GnRH neurones themselves. The underlying signalling pathways responsible for these structural changes are comprised of highly diffusible gaseous molecules, such as endothelial nitric oxide, and paracrine communication processes involving receptors of the erbB tyrosine kinase family, transforming growth factor beta 1 and eicosanoids, such as prostagladin E₂. Some of these molecules, as a result of their ability to diffuse within the median eminence, may also serve as synchronising cues allowing for the occurrence of functionally meaningful episodes of GnRH secretion by coordinating GnRH release from the GnRH neuroendocrine terminals.     

Nuclear Thimet Oligopeptidase is Coexpressed with Oestrogen Receptor α in Hypothalamic Cells and Regulated by Oestradiol in Female Mice

Thimet oligopeptidase (EC 3.4.24.15; also called EP24.15 and TOP; referred to here as TOP) is a neuropeptidase involved in the regulation of several physiological functions including reproduction. Among its substrates is gonadotrophin‐releasing hormone (GnRH), an important hypothalamic hormone that regulates the synthesis and release of oestradiol and facilitates female sexual behaviour. Using immunohistochemistry, we found that TOP is expressed in the nucleus of cells throughout the female mouse brain, and in high levels in steroid‐sensitive regions of the hypothalamus, which is consistent with previous findings in male rats. Furthermore, dual‐label immunofluorescence revealed that TOP and oestrogen receptor α (ERα) coexpress in several reproductively‐relevant brain regions, including the medial preoptic area (mPOA), arcuate nucleus (ARC), ventrolateral portion of the ventromedial hypothalamic nucleus (VMNvl) and the midbrain central grey (MCG). Previous studies in rats have shown that oestradiol decreases hypothalamic TOP levels or activity, possibly potentiating the effects of GnRH. In the present study, analysis by immunohistochemistry revealed that oestradiol decreased TOP immunoreactivity in the VMNvl, whereas no differences were detected in the mPOA, ARC or median eminence. Overall, the present findings indicate that TOP is coexpressed with ERα, and oestradiol regulates TOP expression in a brain region‐specific manner in female mice, providing neuroanatomical evidence that TOP may function in reproductive physiology and/or behaviour.