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.     

Multiple failures in the lutenising hormone surge generating system in GABAB1KO female mice

Female mice lacking GABAB receptors, GABAB1KO, show disrupted oestrous cycles, reduced pregnancies and increased hypothalamic Gnrh1 mRNA expression, whereas anteroventral periventricular/periventricular preoptic nucleus (AVPV/PeN) Kiss1 mRNA was not affected. In the present study, we characterise the important components of the gonadotrophic preovulatory surge, aiming to unravel the origin of this reproductive impairment. In GABAB1KO and wild‐type (WT) females, we determined: (i) hypothalamic oestrogen receptor (ER)α and β and aromatase mRNA and protein expression; (ii) ovulation index and oestrus serum follicle‐stimulating hormone (FSH) and pituitary Gnrh1r expression; (iii) in ovariectomised‐oestradiol valerate‐treated mice, we evaluated ex vivo hypothalamic gonadotrophin‐releasing hormone (GnRH) pulsatility in the presence/absence of kisspeptin (Kiss‐10, constant or pulsatile) and oestradiol (constant); and (iv) in ovariectomised‐oestradiol silastic capsule‐treated mice (proestrous‐like environment), we evaluated morning and evening kisspeptin neurone activation (c‐Fos+) and serum luteinising homrone (LH). In the medial basal hypothalamus of oestrus GABAB1KOs, aromatase and ERα mRNA and protein were increased, whereas ERβ was decreased. In GABAB1KOs, the ovulation index was decreased together with decreased first oestrus serum FSH and increased pituitary Gnrh1r mRNA. Under constant Kiss‐10 stimulation, hypothalamic GnRH pulse frequency did not vary, although GnRH mass/pulse was increased in GABAB1KOs. In WTs, pulsatile Kiss‐10 together with constant oestradiol significantly increased GnRH pulsatility, whereas, in GABAB1KOs, oestradiol alone increased GnRH pulsatility and this was reversed by pulsatile Kiss‐10 addition. In GABAB1KOs AVPV/PeN kisspeptin neurones were similarly activated (c‐Fos+) in the morning and evening, whereas WTs showed the expected, marked evening stimulation. LH correlated with activated kisspeptin cells in WT mice, whereas GABAB1KO mice showed high, similar LH levels both in the morning and evening. Taken together, all of these alterations point to impairment in the trigger of the preovulatory GnRH surge that entails the reproductive alterations described.     

Sexually dimorphic expression of hypothalamic estrogen receptors α and β and Kiss1 in neonatal male and female rats

Release of gonadotropins in adult rodents is sex specific and dependent upon kisspeptin (Kiss1) neurons. This crucial pathway within the hypothalamic-pituitary-gonadal (HPG) axis is profoundly influenced by neonatal estrogens, which induce a male-like phenotype. Classically, estrogen activity is mediated via the estrogen receptors α and β (ERα and ERβ), but the relative roles each plays in organizing the sex-specific ontogeny of kisspeptin signaling pathways remain unresolved. Thus, the present study used in situ hybridization histochemistry (ISHH) to map the temporal and sexually dimorphic neonatal mRNA expression profiles of ERα, ERβ, and Kiss1 in the anterioventral periventricular nucleus (AVPV), medial preoptic area (MPOA), ventromedial nucleus (VMN), and arcuate nucleus (ARC), all regions critical for kisspeptin regulation of gonadotropin secretion. In general, females had higher levels of ERα, in all regions examined, a sex difference that persisted until postnatal day (PND) 19 except in the ARC. Males had significantly more ERβ expression in the AVPV at birth, but this sex difference was lost and then re-emerged on PND 19, with females having more than males. VMN ERβ levels were higher in females until PND 19. Kiss1 was not detectable until PND 11 in the anterior hypothalamus, but expression levels were equivalent at birth in the ARC. By PND 2, ARC ERα and Kiss1 levels were abundant, sexually dimorphic (higher in females), and, respectively, showed a U- and a bell-shaped pattern with age. Sex differences in ARC Kiss1 expression provide evidence that Kiss1 may play a role in the sexual dimorphic organization of the neonatal brain. These detailed profiles of neonatal Kiss1 and ERs mRNA levels will help elucidate the relative roles each plays in the sex-specific, estrogen-dependent organization of gonadotropin signaling pathways.     

Characterization and gonadal hormone regulation of a sexually dimorphic corticotropin-releasing factor receptor 1 cell group

Corticotropin-releasing factor binds with high affinity to CRF receptor 1 (CRFR1) and is implicated in stress-related mood disorders such as anxiety and depression. Using a validated CRFR1-green fluorescent protein (GFP) reporter mouse, our laboratory recently discovered a nucleus of CRFR1 expressing cells that is prominent in the female rostral anteroventral periventricular nucleus (AVPV/PeN), but largely absent in males. This sex difference is present in the early postnatal period and remains dimorphic into adulthood. The present investigation sought to characterize the chemical composition and gonadal hormone regulation of these sexually dimorphic CRFR1 cells using immunohistochemical procedures. We report that CRFR1-GFP-ir cells within the female AVPV/PeN are largely distinct from other dimorphic cell populations (kisspeptin, tyrosine hydroxylase). However, CRFR1-GFP-ir cells within the AVPV/PeN highly co-express estrogen receptor alpha as well as glucocorticoid receptor. A single injection of testosterone propionate or estradiol benzoate on the day of birth completely eliminates the AVPV/PeN sex difference, whereas adult gonadectomy has no effect on CRFR1-GFP cell number. These results indicate that the AVPV/PeN CRFR1 is regulated by perinatal but not adult gonadal hormones. Finally, female AVPV/PeN CRFR1-GFP-ir cells are activated following an acute 30-min restraint stress, as assessed by co-localization of CRFR1-GFP cells with phosphorylated (p) CREB. CRFR1-GFP/pCREB cells were largely absent in the male AVPV/PeN. Together, these data indicate a stress and gonadal hormone responsive nucleus that is unique to females and may contribute to sex-specific stress responses.     

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.     

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.     

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.     

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.     

Sex Differences and the Roles of Sex Steroids in Apoptosis of Sexually Dimorphic Nuclei of the Preoptic Area in Postnatal Rats

The brain contains several sexually dimorphic nuclei that exhibit sex differences with respect to cell number. It is likely that the control of cell number by apoptotic cell death in the developing brain contributes to creating sex differences in cell number in sexually dimorphic nuclei, although the mechanisms responsible for this have not been determined completely. The milieu of sex steroids in the developing brain affects sexual differentiation in the brain. The preoptic region of rats has two sexually dimorphic nuclei. The sexually dimorphic nucleus of the preoptic area (SDN-POA) has more neurones in males, whereas the anteroventral periventricular nucleus (AVPV) has a higher cell density in females. Sex differences in apoptotic cell number arise in the SDN-POA and AVPV of rats in the early postnatal period, and an inverse correlation exists between sex differences in apoptotic cell number and the number of living cells in the mature period. The SDN-POA of postnatal male rats exhibits a higher expression of anti-apoptotic Bcl-2 and lower expression of pro-apoptotic Bax compared to that in females and, as a potential result, apoptotic cell death via caspase-3 activation more frequently occurs in the SDN-POA of females. The patterns of expression of Bcl-2 and Bax in the SDN-POA of postnatal female rats are changed to male-typical ones by treatment with oestrogen, which is normally synthesised from testicular androgen and affects the developing brain in males. In the AVPV of postnatal rats, apoptotic regulation also differs between the sexes, although Bcl-2 expression is increased and Bax expression and caspase-3 activity are decreased in females. The mechanisms of apoptosis possibly contributing to the creation of sex differences in cell number and the roles of sex steroids in apoptosis are discussed.     

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.     

Correlation of Hypothalamic Somatostatin mRNA Expression and Peptide Content with Secretion: Sexual Dimorphism and Differential Regulation by Gonadal Factors

Sex differences in growth hormone (GH) secretion in the rat are thought to be determined, to a large extent, by gonadal steroid-dependent sex differences in somatostatin (SRIH) secretion from neurones in the periventricular nucleus (PeN) which project to the median eminence (ME). The present study aimed to obtain direct evidence for sex differences and gonadal regulation of SRIH release within this pathway and to determine the relationships between SRIH mRNA expression, SRIH peptide content and SRIH secretion in the adult rat. Somatostatin mRNA expression in the PeN and peptide content in both PeN and ME were higher in males than females (P<0.05). However, both basal and 56  m m K⁺-stimulated SRIH release in vitro from hypothalamic explants incorporating the PeN–ME pathway were higher (P<0.01) in females. The gonadectomy of female rats resulted in significantly reduced basal levels of SRIH release equivalent to that of males but had no effect on SRIH mRNA/peptide content or K⁺-stimulated release. In contrast, gonadectomy of male rats reduced SRIH mRNA and peptide contents and elevated K⁺-stimulated secretion (P<0.01) to levels similar to that seen in intact females, without affecting basal release. In summary, these results demonstrate that in the PeN-ME of the adult rat: (1) SRIH mRNA and peptide content is well correlated and sexually dimorphic but dependent on gonadal factors in the male only; (2) SRIH secretion is sexually dimorphic and dependent on gonadal factors; but (3) differences in mRNA/peptide content do not reflect secretory capacity; and (4) gonadal factors differentially modulate SRIH secretory dynamics in males and females.     

Gonadal Steroid Action and Brain Sex Differentiation in the Rat

Gonadal steroids that establish sexually dimorphic characteristics of brain morphology and physiology act at a particular stage of ontogeny. Testosterone secreted by the testes during late gestational and neonatal periods causes significant brain sexual dimorphism in the rat. This results in both sex-specific behaviour and endocrinology in adults. Sexual differentiation may be due to neurogenesis, migration or survival. Each mechanism appears to be uniquely regulated in a site-specific manner. Thus, the volume of an aggregate of neurones in the rat medial preoptic area (POA), termed the sexually dimorphic nucleus of the POA (SDN-POA), is larger in males than in females. The anteroventral periventricular nucleus (AVPV) is packed with neurones containing oestrogen receptor (ER)β in female rats but, in males, ERβ-positive neurones scatter into the more lateral portion of the POA. POA neurones are born up to embryonic days 16–17 and not after parturition. Therefore, neurogenesis is unlikely to contribute to the larger SDN-POA in males. DNA microarray analysis for oestrogen-responsive genes and western blotting demonstrated site-specific regulation of apoptosis- and migration-related genes in the SDN-POA and AVPV.     

Characterisation of arcuate nucleus kisspeptin/neurokinin B neuronal projections and regulation during lactation in the rat

Lactation results in negative energy balance in the rat leading to decreased gonadotrophin-releasing hormone (GnRH) release and anoestrus. Inhibited GnRH release may be a result of decreased stimulatory tone from neuropeptides critical for GnRH neuronal activity, such as kisspeptin (Kiss1) and neurokinin B (NKB). The present study aimed to identify neuronal projections from the colocalised population of Kiss1/NKB cells in the arcuate nucleus (ARH) using double-label immunohistochemistry to determine where this population may directly regulate GnRH neuronal activity. Additionally, the present study further examined lactation-induced changes in the Kiss1 system that could play a role in decreased GnRH release. The colocalised ARH Kiss1/NKB fibres projected primarily to the internal zone of the median eminence (ME) where they were in close proximity to GnRH fibres; however, few Kiss1/NKB fibres from the ARH were seen at the level of GnRH neurones in the preoptic area (POA). Arcuate Kiss1/NKB peptide levels were decreased during lactation consistent with previous mRNA data. Surprisingly, anteroventral periventricular (AVPV) Kiss1 peptide levels were increased, whereas Kiss1 mRNA levels were decreased during lactation, suggesting active inhibition of peptide release. These findings indicate ARH Kiss1/NKB and AVPV Kiss1 appear to be inhibited during lactation, which may contribute to decreased GnRH release and subsequent reproductive dysfunction. Furthermore, the absence of a strong ARH Kiss1/NKB projection to the POA suggests regulation of GnRH by this population occurs primarily at the ME level via local projections.     

Oestrogen‐Independent Circadian Clock Gene Expression in the Anteroventral Periventricular Nucleus in Female Rats: Possible Role as an Integrator for Circadian and Ovarian Signals Timing the Luteinising Hormone Surge

Periodic ovulation in rats, mice and hamsters is the result of a surge in luteinising hormone (LH) that depends on circadian gating signals emerging from the master circadian clock within the suprachiasmatic nucleus (SCN) and rising ovarian oestrogen levels. These two signals converge into the anteroventral periventricular nucleus (AVPV) and lead to the release of kisspeptin, which is responsible for surges of gonadotrophin‐releasing hormone and, in turn, of LH release. How the AVPV integrates circadian and reproductive signals remains unclear. In the present study, we show that the female rat AVPV itself shows circadian oscillations in the expression of the clock genes PER1 and BMAL1, which lie at the core circadian clockwork of mammals. In ovariectomised females treated with oestradiol (E₂), these oscillations are in synchrony with the AVPV rhythmic expression of the KISS1 gene and the gene that codes for the arginine‐vasopressin (AVP) receptor AVPr1a. Although clock gene oscillations are independent of oestrogen levels, circadian expression of Kiss1 and Avpr1a (also referred to as V1a) mRNA is blunted and absent, respectively, in ovariectomised animals without E₂ replacement. Because AVP is considered to be a critical SCN transmitter to gate the LH surge, our data suggest that there is a circadian oscillator located in the AVPV, and that such a putative oscillator could, in an oestrogen‐dependent manner, time the sensitivity to circadian signals emerging from the SCN and the release of kisspeptin.     

Enhancement of the luteinising hormone surge by male olfactory signals is associated with anteroventral periventricular Kiss1 cell activation in female rats

Olfactory stimuli play an important role in regulating reproductive functions in mammals. The present study investigated the effect of olfactory signals derived from male rats on kisspeptin neuronal activity and luteinising hormone (LH) secretion in female rats. Wistar‐Imamichi strain female rats were ovariectomised (OVX) and implanted with preovulatory levels of 17β‐oestradiol (E₂). OVX+E₂ rats were killed 1 hour after exposure to either: clean bedding, female‐soiled bedding or male‐soiled bedding. Dual staining for Kiss1 mRNA in situ hybridisation and c‐Fos immunohistochemistry revealed that the numbers of Kiss1‐expressing cells and c‐Fos‐immunopositive Kiss1‐expressing cells in the anteroventral periventricular nucleus (AVPV) were significantly higher in OVX+E₂ rats exposed to male‐soiled bedding than those of the other groups. No significant difference was found with respect to the number of c‐Fos‐immunopositive Kiss1‐expressing cells in the arcuate nucleus and c‐Fos‐immunopositive Gnrh1‐expressing cells between the groups. The number of c‐Fos‐immunopositive cells was also significantly higher in the limbic system consisting of several nuclei, such as the bed nucleus of the stria terminalis, the cortical amygdala and the medial amygdala, in OVX+E₂ rats exposed to male‐soiled bedding than the other groups. OVX+E₂ rats exposed to male‐soiled bedding showed apparent LH surges, and the peak of the LH surge and area under the curve of LH concentrations in the OVX+E₂ group were significantly higher than those of the other two groups. These results suggest that olfactory signals derived from male rats activate AVPV kisspeptin neurones, likely via the limbic system, resulting in enhancement of the peak of the LH surge in female rats. Taken together, the results of the present study suggests that AVPV kisspeptin neurones are a target of olfactory signals to modulate LH release in female rats.     

Lack of Pulse and Surge Modes and Glutamatergic Stimulation of Luteinising Hormone Release in Kiss1 Knockout Rats

Kisspeptin, encoded by the Kiss1 gene, has attracted attention as a key candidate neuropeptide in controlling puberty and reproduction via regulation of gonadotrophin‐releasing hormone (GnRH) secretion in mammals. Pioneer studies with Kiss1 or its cognate receptor Gpr54 knockout (KO) mice showed the indispensable role of kisspeptin‐GPR54 signalling in the control of animal reproduction, although detailed analyses of gonadotrophin secretion, especially pulsatile and surge‐mode of luteinising hormone (LH) secretion, were limited. Thus, in the present study, we have generated Kiss1 KO rats aiming to evaluate a key role of kisspeptin in governing reproduction via pulse and surge modes of GnRH/LH secretion. Kiss1 KO male and female rats showed a complete suppression of pulsatile LH secretion, which is responsible for folliculogenesis and spermatogenesis, and an absence of puberty and atrophic gonads. Kiss1 KO female rats showed no spontaneous LH/follicle‐stimulating hormone surge and an oestrogen‐induced LH surge, suggesting that the GnRH surge generation system, which is responsible for ovulation, does not function without kisspeptin. Furthermore, challenge of major stimulatory neurotransmitters, such as monosodium glutamate, NMDA and norepinephrine, failed to stimulate LH secretion in Kiss1 KO rats, albeit they stimulated LH release in wild‐type controls. Taken together, the results of the present study confirm that kisspeptin plays an indispensable role in generating two modes (pulse and surge) of GnRH/gonadotrophin secretion to regulate puberty onset and normal reproductive performance. In addition, the present study suggests that kisspeptin neurones play a critical role as a hub integrating major stimulatory neural inputs to GnRH neurones, using newly established Kiss1 KO rats, which serve as a useful model for detailed analysis of hormonal profiles.