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.     

Time‐of‐day‐dependent sensitivity of the reproductive axis to RFamide‐related peptide‐3 inhibition in female Syrian hamsters

In spontaneously ovulating rodent species, the timing of the luteinising hormone (LH) surge is controlled by the master circadian pacemaker in the suprachiasmatic nucleus (SCN). The SCN initiates the LH surge via the coordinated control of two opposing neuropeptidergic systems that lie upstream of the gonadotrophin‐releasing hormone (GnRH) neuronal system: the stimulatory peptide, kisspeptin, and the inhibitory peptide, RFamide‐related peptide‐3 (RFRP‐3; the mammalian orthologue of avian gonadotrophin‐inhibitory hormone [GnIH]). We have previously shown that the GnRH system exhibits time‐dependent sensitivity to kisspeptin stimulation, further contributing to the precise timing of the LH surge. To examine whether this time‐dependent sensitivity of the GnRH system is unique to kisspeptin or a more common mechanism of regulatory control, we explored daily changes in the response of the GnRH system to RFRP‐3 inhibition. Female Syrian hamsters were ovariectomised to eliminate oestradiol (E₂)‐negative‐feedback and RFRP‐3 or saline was centrally administered in the morning or late afternoon. LH concentrations and Lhβ mRNA expression did not differ between morning RFRP‐3‐and saline‐treated groups, although they were markedly suppressed by RFRP‐3 administration in the afternoon. However, RFRP‐3 inhibition of circulating LH at the time of the surge does not appear to act via the GnRH system because no differences in medial preoptic area Gnrh or RFRP‐3 receptor Gpr147 mRNA expression were observed. Rather, RFRP‐3 suppressed arcuate nucleus Kiss1 mRNA expression and potentially impacted pituitary gonadotrophs directly. Taken together, these findings reveal time‐dependent responsiveness of the reproductive axis to RFRP‐3 inhibition, possibly via variation in the sensitivity of arcuate nucleus kisspeptin neurones to this neuropeptide.     

Characterisation of Kiss1r (Gpr54)‐Expressing Neurones in the Arcuate Nucleus of the Female Rat Hypothalamus

Kisspeptin is essential in reproduction and acts by stimulating neurones expressing gonadotrophin‐releasing hormone (GnRH). Recent studies suggest that kisspeptin has multiple roles in the modulation of neuronal circuits in systems outside the hypothalamic‐pituitary‐gonadal axis. Our recent research using in situ hybridisation (ISH) clarified the histological distribution of Kiss1r (Gpr54)‐expressing neurones in the rat brain that were presumed to be putative targets of kisspeptin. The arcuate nucleus (ARN) of the hypothalamus is one of the brain regions in which Kiss1r expression in non‐GnRH neurones is prominent. However, the characteristics of Kiss1r‐expressing neurones in the ARN remain unclear. The present study aimed to determine the neurochemical characteristics of Kiss1r‐expressing neurones in the ARN using ISH and immunofluorescence. We revealed that the majority (approximately 63%) of Kiss1r‐expressing neurones in the ARN were pro‐opiomelanocortin (POMC) neurones, which have an anorexic effect in mammals. Additionally, a few Kiss1r‐expressing neurones in the dorsal ARN are tuberoinfundibular dopamine (TIDA) neurones, which control milk production by inhibiting prolactin secretion from the anterior pituitary. TIDA neurones showed a relatively weak Kiss1r ISH signal compared to POMC neurones, as well as low co‐expression of Kiss1r (approximately 15%). We also examined the expression of Kiss1r in neuropeptide Y and kisspeptin neurones, which are reported to arise from POMC‐expressing progenitor cells during development. However, the vast majority of neuropeptide Y and kisspeptin neurones in the ARN did not express Kiss1r. These results suggest that kisspeptin may directly regulate energy homeostasis and milk production by modulating the activity of POMC and TIDA neurones, respectively. Our results provide an insight into the wide variety of roles that kisspeptin plays in homeostatic and neuroendocrine functions.     

Reproductive status‐dependent kisspeptin and RFamide‐related peptide (Rfrp) gene expression in female Damaraland mole‐rats

Damaraland mole rats (Fukomys damarensis) are cooperatively breeding, subterranean mammals that exhibit a high reproductive skew. Reproduction is monopolised by the dominant female of the group, whereas subordinates are physiologically suppressed to the extent that they are anovulatory. In these latter animals, it is assumed that normal gonadotropin‐releasing hormone secretion from the hypothalamus is disrupted. The RFamide peptides kisspeptin (Kiss1) and RFamide‐related peptide‐3 (RFRP‐3) are considered as potent regulators of gonadotropin release. To assess whether these neuropeptides are involved in the mechanism of reproductive suppression, we investigated the distribution and gene expression of Kiss1 and Rfrp by means of in situ hybridisation in wild‐caught female Damaraland mole‐rats with different reproductive status. In both reproductive phenotypes, substantial Kiss1 expression was found in the arcuate nucleus and only few Kiss1‐expressing cells were detected in the anteroventral periventricular nucleus (AVPV), potentially as a result of low circulating oestradiol concentrations in breeding and nonbreeding females. Rfrp gene expression occurred in the dorsomedial nucleus, the paraventricular nucleus and the periventricular nucleus. While in female breeders and nonbreeders, plasma oestradiol levels were low and not significantly different, quantification of the hybridisation signal for both genes revealed significant differences in relation to reproductive status. Reproductively active females had more Kiss1‐expressing cells and a higher number of silver grains per cell in the arcuate nucleus compared to nonreproductive females. This difference was most pronounced in the caudal part of the nucleus. No such differences were found in the AVPV. Furthermore, breeding status was associated with a reduced number of Rfrp‐expressing cells in the anterior hypothalamus. This reproductive status‐dependent expression pattern of Kiss1 and Rfrp suggests that both neuropeptides play a role in the regulation of reproduction in Damaraland mole‐rats. Enhanced long‐term negative feedback effects of oestradiol could be responsible for the lower Kiss1 expression in the arcuate nucleus of reproductively suppressed females.     

Gonadal and Nongonadal Regulation of Sex Differences in Hypothalamic Kiss1 Neurones

The brains of males and females differ anatomically and physiologically, including sex differences in neurone size or number, synapse morphology and specific patterns of gene expression. Brain sex differences may underlie critical sex differences in physiology or behaviour, including several aspects of reproduction, such as the timing of sexual maturation (earlier in females than males) and the ability to generate a preovulatory gonadotrophin surge (in females only). The reproductive axis is controlled by afferent pathways that converge upon forebrain gonadotrophin‐releasing hormone (GnRH) neurones, but GnRH neurones are not sexually dimorphic. Although most reproductive sex differences probably reflect sex differences in the upstream circuits and factors that regulate GnRH secretion, the key sexually‐dimorphic factors that influence reproductive status have remained poorly defined. The recently‐identified neuropeptide kisspeptin, encoded by the Kiss1 gene, is an important regulator of GnRH secretion, and Kiss1 neurones in rodents are sexually dimorphic in specific hypothalamic populations, including the anteroventral periventricular nucleus–periventricular nucleus continuum (AVPV/PeN) and the arcuate nucleus (ARC). In the adult AVPV/PeN, Kiss1 neurones are more abundant in females than males, representing a sex difference that is regulated by oestradiol signalling during critical periods of postnatal and pubertal development. By contrast, Kiss1 neurones in the ARC are not sexually differentiated in adult rodents but, in mice, the regulation of ARC Kiss1 cells by gonadal hormone‐independent factors is sexually dimorphic during prepubertal development. These various sex differences in hypothalamic Kiss1 neurones may relate to known sex differences in reproductive physiology, such as puberty onset and positive feedback.     

Photoperiodic Co‐Regulation of Kisspeptin,Neurokinin B and Dynorphin in the Hypothalamus of a Seasonal Rodent

In many species, sexual activity varies on a seasonal basis. Kisspeptin (Kp), a hypothalamic neuropeptide acting as a strong activator of gonadotrophin‐releasing hormone neurones, plays a critical role in this adaptive process. Recent studies report that two other neuropeptides, namely neurokinin B (NKB) and dynorphin (DYN), are co‐expressed with Kp (and therefore termed KNDy neurones) in the arcuate nucleus and that these peptides are also considered to influence GnRH secretion. The present study aimed to establish whether hypothalamic NKB and DYN expression is photoperiod‐dependent in a seasonal rodent, the Syrian hamster, which exhibits robust seasonal rhythms in reproductive activity. The majority of Kp neurones in the arcuate nucleus co‐express NKB and DYN and the expression of all three peptides is decreased under a short (compared to long) photoperiod, leading to a 60% decrease in the number of KNDy neurones under photo‐inhibitory conditions. In seasonal rodents, RFamide‐related peptide (RFRP) neurones of the dorsomedial hypothalamus are also critical for seasonal reproduction. Interestingly, NKB and DYN are also expressed in the dorsomedial hypothalamus but do not co‐localise with RFRP‐immunoreactive neurones, and the expression of both NKB and DYN is higher under a short photoperiod, which is opposite to the short‐day inhibition of RFRP expression. In conclusion, the present study shows that NKB and DYN display different photoperiodic variations in the Syrian hamster hypothalamus. In the arcuate nucleus, NKB and DYN, together with Kp, are down‐regulated under a short photoperiod, whereas, in the dorsomedial hypothalamus, NKB and DYN are up‐regulated under a short photoperiod.     

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.     

Surge‐Like Luteinising Hormone Secretion Induced by Retrochiasmatic Area NK3R Activation is Mediated Primarily by Arcuate Kisspeptin Neurones in the Ewe

The neuropeptides neurokinin B (NKB) and kisspeptin are potent stimulators of gonadotrophin‐releasing hormone (GnRH)/luteinsing hormone (LH) secretion and are essential for human fertility. We have recently demonstrated that selective activation of NKB receptors (NK3R) within the retrochiasmatic area (RCh) and the preoptic area (POA) triggers surge‐like LH secretion in ovary‐intact ewes, whereas blockade of RCh NK3R suppresses oestradiol‐induced LH surges in ovariectomised ewes. Although these data suggest that NKB signalling within these regions of the hypothalamus mediates the positive‐feedback effects of oestradiol on LH secretion, the pathway through which it stimulates GnRH/LH secretion remains unclear. We proposed that the action of NKB on RCh neurones drives the LH surge by stimulating kisspeptin‐induced GnRH secretion. To test this hypothesis, we quantified the activation of the preoptic/hypothalamic populations of kisspeptin neurones in response to POA or RCh administration of senktide by dual‐label immunohistochemical detection of kisspeptin and c‐Fos (i.e. marker of neuronal activation). We then administered the NK3R agonist, senktide, into the RCh of ewes in the follicular phase of the oestrous cycle and conducted frequent blood sampling during intracerebroventricular infusion of the kisspeptin receptor antagonist Kp‐271 or saline. Our results show that the surge‐like secretion of LH induced by RCh senktide administration coincided with a dramatic increase in c‐Fos expression within arcuate nucleus (ARC) kisspeptin neurones, and was completely blocked by Kp‐271 infusion. We substantiate these data with evidence of direct projections of RCh neurones to ARC kisspeptin neurones. Thus, NKB‐responsive neurones in the RCh act to stimulate GnRH secretion by inducing kisspeptin release from KNDy neurones.     

Visualisation of Kiss1 Neurone Distribution Using a Kiss1‐CRE Transgenic Mouse

Kisspeptin neuropeptides are encoded by the Kiss1 gene and play a critical role in the regulation of the mammalian reproductive axis. Kiss1 neurones are found in two locations in the rodent hypothalamus: one in the arcuate nucleus (ARC) and another in the RP3V region, which includes the anteroventral periventricular nucleus (AVPV). Detailed mapping of the fibre distribution of Kiss1 neurones will help with our understanding of the action of these neurones in other regions of the brain. We have generated a transgenic mouse in which the Kiss1 coding region is disrupted by a CRE‐GFP transgene so that expression of the CRE recombinase protein is driven from the Kiss1 promoter. As expected, mutant mice of both sexes are sterile with hypogonadotrophic hypogonadism and do not show the normal rise in luteinising hormone after gonadectomy. Mutant female mice do not develop mature Graafian follicles or form corpora lutea consistent with ovulatory failure. Mutant male mice have low blood testosterone levels and impaired spermatogenesis beyond the meiosis stage. Breeding Kiss‐CRE heterozygous mice with CRE‐activated tdTomato reporter mice allows fluorescence visualisation of Kiss1 neurones in brain slices. Approximately 80‐90% of tdTomato positive neurones in the ARC were co‐labelled with kisspeptin and expression of tdTomato in the AVPV region was sexually dimorphic, with higher expression in females than males. A small number of tdTomato‐labelled neurones was also found in other locations, including the lateral septum, the anterodorsal preoptic nucleus, the amygdala, the dorsomedial and ventromedial hypothalamic nuclei, the periaquaductal grey, and the mammillary nucleus. Three dimensional visualisation of Kiss1 neurones and fibres by CLARITY processing of whole brains showed an increase in ARC expression during puberty and higher numbers of Kiss1 neurones in the caudal region of the ARC compared to the rostral region. ARC Kiss1 neurones sent fibre projections to several hypothalamic regions, including rostrally to the periventricular and pre‐optic areas and to the lateral hypothalamus.     

Evidence of involvement of neurone‐glia/neurone‐neurone communications via gap junctions in synchronised activity of KNDy neurones

Pulsatile secretion of gonadotrophin‐releasing hormone (GnRH)/luteinising hormone is indispensable for the onset of puberty and reproductive activities at adulthood in mammalian species. A cohort of neurones expressing three neuropeptides, namely kisspeptin, encoded by the Kiss1 gene, neurokinin B (NKB) and dynorphin A, localised in the hypothalamic arcuate nucleus (ARC), so‐called KNDy neurones, comprises a putative intrinsic source of the GnRH pulse generator. Synchronous activity among KNDy neurones is considered to be required for pulsatile GnRH secretion. It has been reported that gap junctions play a key role in synchronising electrical activity in the central nervous system. Thus, we hypothesised that gap junctions are involved in the synchronised activities of KNDy neurones, which is induced by NKB‐NK3R signalling. We determined the role of NKB‐NK3R signalling in Ca²⁺ oscillation (an indicator of neuronal activities) of KNDy neurones and its synchronisation mechanism among KNDy neurones. Senktide, a selective agonist for NK3R, increased the frequency of Ca²⁺ oscillations in cultured Kiss1‐GFP cells collected from the mediobasal hypothalamus of the foetal Kiss1‐green fluorescent protein (GFP) mice. The senktide‐induced Ca²⁺ oscillations were synchronised in the Kiss1‐GFP and neighbouring glial cells. Confocal microscopy analysis of these cells, which have shown synchronised Ca²⁺ oscillations, revealed close contacts between Kiss1‐GFP cells, as well as between Kiss1‐GFP cells and glial cells. Dye coupling experiments suggest cell‐to‐cell communication through gap junctions between Kiss1‐GFP cells and neighbouring glial cells. Connexin‐26 and ‐37 mRNA were found in isolated ARC Kiss1 cells taken from adult female Kiss1‐GFP transgenic mice. Furthermore, 18β‐glycyrrhetinic acids and mefloquine, which are gap junction inhibitors, attenuated senktide‐induced Ca²⁺ oscillations in Kiss1‐GFP cells. Taken together, these results suggest that NKB‐NK3R signalling enhances synchronised activities among neighbouring KNDy neurones, and that both neurone‐neurone and neurone‐glia communications via gap junctions possibly contribute to synchronised activities among KNDy neurones.     

The Effects of Neurokinin B upon Gonadotrophin Release in Male Rodents

Growing evidence suggests the tachykinin neurokinin B (NKB) may modulate gonadotrophin secretion and play a role in sex‐steroid feedback within the reproductive axis. NKB signalling has recently been identified as being necessary for normal human reproductive function, although the precise mechanisms underpinning this role remain to be established. We have used rodents to explore further the role of NKB within the reproductive axis. In particular, we have studied its interactions with kisspeptin, a neuropeptide essential for reproductive function in rodent and human with close anatomical links to NKB within the hypothalamus. Intraperitoneal administration of NKB (50 nmol) to male mice had no effect on circulating luteinsing hormone (LH) levels and, although i.p. kisspeptin (15 nmol) increased LH five‐fold, co‐administration of NKB and kisspeptin was indistinguishable from kisspeptin alone. Intracerebroventricular administration of NKB (10 nmol) to male mice also had no effect on LH levels, with 1 nmol kisspeptin i.c.v. significantly increasing LH compared to control (0.37 ± 0.18 versus 5.11 ± 0.28 ng/ml, respectively). Interestingly, i.c.v. co‐administration of NKB and kisspeptin caused a significant increase in LH concentrations compared to kisspeptin alone (8.96 ± 1.82 versus 5.11 ± 0.28 ng/ml respectively). We used hypothalamic explants from rats to assess the effect of NKB on gonadotrpohin‐releasing hormone (GnRH) secretion ex vivo. Doses of NKB up to 1000 nm failed to stimulate GnRH secretion, whereas 100 nm kisspeptin robustly increased GnRH secretion. Of note, co‐administration of NKB with kisspeptin abrogated the effect of kisspeptin, producing no GnRH release above basal state. Finally, we analysed the expression of Tac2/Tacr3 (genes encoding NKB and NK3R, respectively) within the arcuate nucleus in different nutritional states. After a 48‐h fast, the expression of both Tac2 and Tacr3 showed a significant increase, in contrast to levels of Kiss1 and Kiss1r mRNA, which remained unchanged. In male rodent models, NKB and kisspeptin have different effects upon gonadotrophin release and appear to interact in a complex manner.     

The Content of Thyroid Hormone Receptor α in Ewe Kisspeptin Neurones is not Season‐Dependent

Seasonal reproduction is grounded in several mechanisms, among which are plasticity in both hormone synthesis and neuronal networks. Increased daylength on long days (LD) translates into local tri‐iodothyronin (T3) production in the mediobasal hypothalamus that will enable the transition to the anoestrus season in sheep. The photoperiod also strongly affects the content of kisspeptin (Kiss), a hypothalamic neuropeptide exerting a potent stimulatory effect on gonadotrophin‐releasing hormone release. Our hypothesis was that T3 directly inhibits Kiss release during LD. Using double immunocytochemistry, we first searched for coexpression of thyroid hormone receptor (THR)α in Kiss neurones in ewes with an active or inactive gonadotrophic axis. In both the preoptic area and the arcuate nucleus, most Kiss neurones were labelled by THR antibody under both physiological/photoperiodic conditions. These results suggest thyroid hormones may affect Kiss synthesis and release all through the year. We then attempted to assess the influence of T3 on Kiss content in hypothalamic explants sampled from ewes with an active gonadotrophic axis. Kiss produced by hypothalamic explants cultured with different doses of T3 (300 or 600 pg) and subjected to different times of incubation (2 or 24 h) was measured. No significant effects of T3 on Kiss tissular content were observed for the two doses of T3 and for the two incubation times. In light of these findings, potential reasons for the divergent effects of thyroid hormones on Kiss content are discussed. Our data emphasise that the effects of thyroid hormone on Kiss synthesis are not one‐sided and may affect a wide range of functions.     

Kisspeptin signalling in the brain: steroid regulation in the rodent and ewe

The Kiss1 gene encodes a family of peptides called kisspeptins, which are the natural ligands for the receptor GPR54. In humans and mice, inactivating mutations of GPR54 results in hypogonadotropic hypogonadism, indicating that kisspeptins play a vital role in the regulation of GnRH secretion. In many species, centrally administered kisspeptins stimulate gonadotrophin secretion in a GnRH-dependant manner. Moreover, virtually all GnRH neurons coexpress GPR54. In the hypothalamus, the vast majority of kisspeptin producing cells also express sex steroid receptors, particularly estrogen receptor alpha. Thus, sex steroids are able to directly regulate the expression of Kiss1 mRNA, implicating kisspeptins as the ‘missing link’ between sex steroid feedback and GnRH secretion. Kiss1-expressing cells are localised to various regions of the forebrain in rodents, primates and sheep. In the arcuate nucleus (ARC) of the rodent and the ewe, sex steroids inhibit the expression of Kiss1 mRNA, suggesting that the kisspeptin secreting neurons here are the conduit for the negative feedback regulation of GnRH secretion. However, in the rodent anteroventral periventricular nucleus (AVPV), sex steroids induce the expression of Kiss1, implying that these kisspeptin neurons play a role in the positive feedback regulation of GnRH secretion. In sheep, there are no Kiss1 neurons in the AVPV and Kiss1 mRNA expression in the ARC is stimulated immediately prior to the preovulatory GnRH/luteinising hormone surge. Thus, kisspeptin neurons in the ARC of the ewe appear well placed to play a role in the negative and positive feedback regulation of GnRH exerted by sex steroids.     

Hypothalamic Galanin‐Like Peptide Rescues the Onset of Puberty in Food‐Restricted Weanling Rats

Galanin‐like peptide (GALP) is a known mediator of metabolism and reproduction; however, the role that GALP plays in the onset of puberty is unknown. First, we tested the hypothesis that central GALP administration could rescue puberty in food‐restricted weanling rats. GALP treatment in food‐restricted rats of both sexes rescued the timing of the onset of puberty to that seen in ad lib. fed controls. Second, we tested whether GALP translation knocked‐down in ad lib. fed, prepubertal rats would alter the timing of puberty. Knock‐down females, but not males, showed a significant (P < 0.01) delay in the onset of puberty compared to controls. Third, we sought evidence that the role of GALP in pubertal onset is mediated by the kisspeptin system. In situ hybridisation analyses showed a significant (P < 0.01) reduction in Kiss1 mRNA within the hypothalamic arcuate nucleus in food‐restricted rats compared to ad lib. fed controls and this reduction was prevented with i.c.v. GALP administration. Furthermore, analyses of Fos‐immunoreactivity (‐IR) after i.c.v. GALP treatment did not elicit Fos‐IR within any kisspeptin neurones, nor are GALP and kisspeptin peptides or mRNA colocalised. These data demonstrate that hypothalamic GALP infusion maintained the onset of puberty in food‐restricted weanling rats, although probably not via direct innervation of kisspeptin neurones.     

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.     

Chronic Oestradiol Reduces the Dendritic Spine Density of KNDy (Kisspeptin/Neurokinin B/Dynorphin) Neurones in the Arcuate Nucleus of Ovariectomised Tac2‐Enhanced Green Fluorescent Protein Transgenic Mice

Neurones in the arcuate nucleus that express neurokinin B (NKB), kisspeptin and dynorphin (KNDy) play an important role in the reproductive axis. Oestradiol modulates the gene expression and somatic size of these neurones, although there is limited information available about whether their dendritic structure, a correlate of cellular plasticity, is altered by oestrogens. In the present study, we investigated the morphology of KNDy neurones by filling fluorescent neurones in the arcuate nucleus of Tac2‐enhanced green fluorescent protein (EGFP) transgenic mice with biocytin. Filled neurones from ovariectomised (OVX) or OVX plus 17β‐oestradiol (E₂)‐treated mice were visualised with anti‐biotin immunohistochemistry and reconstructed in three dimensions with computer‐assisted microscopy. KNDy neurones exhibited two primary dendrites, each with a few branches confined to the arcuate nucleus. Quantitative analysis revealed that E₂ treatment of OVX mice decreased the cell size and dendritic spine density of KNDy neurones. The axons of KNDy neurones originated from the cell body or proximal dendrite and gave rise to local branches that appeared to terminate within the arcuate nucleus. Numerous terminal boutons were also visualised within the ependymal layer of the third ventricle adjacent to the arcuate nucleus. Axonal branches also projected to the adjacent median eminence and exited the arcuate nucleus. Confocal microscopy revealed close apposition of EGFP and gonadotrophin‐releasing hormone‐immunoreactive fibres within the median eminence and confirmed the presence of KNDy axon terminals in the ependymal layer of the third ventricle. The axonal branching pattern of KNDy neurones suggests that a single KNDy neurone could influence multiple arcuate neurones, tanycytes in the wall of the third ventricle, axon terminals in the median eminence and numerous areas outside of the arcuate nucleus. In parallel with its inhibitory effects on electrical excitability, E₂ treatment of OVX Tac2‐EGFP mice induces structural changes in the somata and dendrites of KNDy neurones.