Rostro-caudal maturation of glial cells in the accessory olfactory system during development: involvement in outgrowth of GnRH neurites.

During mammalian embryonic development, GnRH neurones differentiate from the nasal placode and migrate through the nasal septum towards the forebrain. We previously showed that a category of glial cells, the olfactory ensheathing cells (OEC), forms the microenvironment of migrating GnRH neurones. Here, to characterize the quantitative and qualitative importance of this glial, we investigated the spatiotemporal maturation of glial cells in situ and the role of maturing glia in GnRH neurones development ex vivo. More than 90% of migrating GnRH neurones were found to be associated with glial cells. There was no change in the cellular microenvironment of GnRH neurones in the regions crossed during embryonic development as glial cells formed the main microenvironment of these neurones (53.4%). However, the phenotype of OEC associated with GnRH neurones changed across regions. The OEC progenitors immunoreactive to brain lipid binding protein formed the microenvironment of migrating GnRH neurones from the vomeronasal organ to the telencephalon and were also present in the diencephalon. However, during GnRH neurone migration, maturation of OEC to [GFAP+] state (glial fibrillary acid protein) was only observed in the nasal septum. Inducing depletion of OEC in maturation, using transgenic mice expressing herpes simplex virus thymidine kinase driven by the GFAP promoter, had no impact on neurogenesis or on triggering GnRH neurones migration in nasal explant culture. Nevertheless, depletion of [GFAP+] cells decreased GnRH neurites outgrowth by 57.4%. This study suggests that specific maturation of OEC in the nasal septum plays a role in morphological differentiation of GnRH neurones.

Elevated body weight gain during the juvenile period alters neuropeptide Y-gonadotropin-releasing hormone circuitry in prepubertal heifers.

Increased body weight (BW) gain during the juvenile period leads to early maturation of the reproductive neuroendocrine system. We investigated whether a nutritional regimen that advances the onset of puberty leads to alterations in the hypothalamic neuropeptide Y (NPY) circuitry that are permissive for enhanced gonadotropin-releasing hormone (GnRH) secretion. It was hypothesized that NPY mRNA and NPY projections to GnRH and kisspeptin neurons are reduced in heifers that gain BW at an accelerated rate, compared with a lower one, during the juvenile period. Heifers were weaned at approximately 4 mo of age and fed diets to promote relatively low (0.5 kg/day; low gain [LG]) or high (1.0 kg/day; high gain [HG]) rates of BW gain until 8.5 mo of age. Heifers that gained BW at a higher rate exhibited greater circulating concentrations of leptin and reduced overall NPY expression in the arcuate nucleus. The proportion of GnRH neurons in close apposition to NPY fibers and the magnitude of NPY projections to GnRH neurons located in the mediobasal hypothalamus were reduced in HG heifers. However, no differences in NPY projections to kisspeptin neurons in the arcuate nucleus were detected between HG and LG heifers. Results indicate that a reduction in NPY innervation of GnRH neurons, particularly at the level of the mediobasal hypothalamus, occurs in response to elevated BW gain during the juvenile period. This functional plasticity may facilitate early onset of puberty in heifers.

Acute injection and chronic perfusion of kisspeptin elicit gonadotropins release but fail to trigger ovulation in the mare.

Kisspeptin has emerged as the most potent gonadotropin-releasing hormone (GnRH) secretagogue and appears to represent the penultimate step in the central control of reproduction. In the sheep, we showed that kisspeptin could be used to manipulate gonadotropin secretion and control ovulation. Prompted by these results, we decided to investigate whether kisspeptin could be used as an ovulation-inducing agent in another photoperiodic domestic mammal, the horse. Equine kisspeptin-10 (eKp10) was administered intravenously as bolus injections or short- to long-term perfusions to Welsh pony mares, either during the anestrus season or at various stages of the cycle during the breeding season. In all the experimental conditions, eKp10 reliably increased peripheral concentrations of both luteinizing hormone and follicle-stimulating hormone. The nature of the response to eKp10 was consistent across experimental conditions and physiological states: the increase in gonadotropins was always rapid and essentially transient even when eKp10 was perfused for prolonged periods. Furthermore, eKp10 consistently failed to induce ovulation in the mare. To gain insights into the underlying mechanisms, we used acute injections or perfusions of GnRH. We also cloned the equine orthologues of the kisspeptin precursor and Kiss1r; this was justified by the facts that the current equine genome assembly predicted an amino acid difference between eKp10 and Kp10 in other species while an equine orthologue for Kiss1r was missing altogether. In light of these findings, potential reasons for the divergence in the response to kisspeptin between ewe and mare are discussed. Our data highlight that kisspeptin is not a universal ovulation-inducing agent.

Hypothalamic distribution, adenohypophyseal receptor expression, and ligand functionality of RFamide-related peptide 3 in the mare during the breeding and nonbreeding seasons.

RFamide-related peptide 3 (RFRP3), the mammalian homologue of avian gonadotropin-inhibitory hormone, has been shown to negatively regulate the secretion of LH and may contribute to reproductive seasonality in some species. Herein, we examined the presence and potential role of the RFRP3-signaling system in regulating LH secretion in the mare during the breeding and nonbreeding seasons. Hypothalamic NPVF mRNA (the precursor mRNA for RFRP3) was detected at the level of the dorsomedial nucleus and paraventricular nucleus, but expression did not change with season. A greater number of RFRP3-expressing cells was observed throughout the rostral-caudal extension of the dorsomedial nucleus. Furthermore, adenohypophyseal expression of the RFRP3 receptor (NPFFR1) during the winter anovulatory season did not differ from that during either the follicular or luteal phases of the estrous cycle. When tested in primary adenohypophyseal cell culture or in vivo during both the breeding and nonbreeding seasons, neither equine nor ovine peptide sequences for RFRP3 suppressed basal or GnRH-mediated release of LH. However, infusion of RF9, an RFRP3 receptor-signaling antagonist, into seasonally anovulatory mares induced a robust increase in secretion of LH both before and following continuous treatment with GnRH. The results indicate that the cellular machinery associated with RFRP3 function is present in the equine hypothalamus and adenohypophysis. However, evidence for functionality of the RFRP3-signaling network was only obvious when an antagonist RF9 was employed. Because GnRH-induced release of LH was not affected by RF9, its actions may occur upstream from the gonadotrope to stimulate or disinhibit secretion of GnRH.

Gonadotropin-releasing hormone neurones innervate kisspeptin neurones in the female mouse brain.

Kisspeptin (KP) neurones in the rostral periventricular area of the third ventricle (RP3V) and arcuate nucleus (Arc) are important elements in the neuronal circuitry regulating gonadotropin-releasing hormone (GnRH) secretion. KP and co-synthesised neuropeptides/neurotransmitters act directly on GnRH perikarya and processes. GnRH neurones not only form the final output pathway regulating the reproductive functions of the anterior pituitary gland, but also provide neuronal input to sites within the hypothalamus. The current double-label immunohistochemical studies investigated whether GnRH-immunoreactive (IR) projections to the RP3V and/or Arc establish morphological connections with KP-IR neurones at these sites. To optimise visualisation of KP immunoreactivity in, respectively, the RP3V and Arc, ovariectomised (OVX) oestrogen-treated and OVX oil-treated female mice were studied. Confocal laser microscopic analysis of immunofluorescent specimens revealed GnRH-IR axon varicosities in apposition to approximately 25% of the KP-IR neurones in the RP3V and 50% of the KP-IR neurones in the Arc. At the ultrastructural level, GnRH-IR neurones were seen to establish asymmetric synaptic contacts, which usually reflect excitatory neurotransmission, with KP-IR neurones in both the RP3V and Arc. Together with previous data, these findings indicate reciprocal connectivity between both of the KP cell populations and the GnRH neuronal system. The functional significance of the GnRH-IR input to the two separate KP cell populations requires electrophysiological investigation.

Kisspeptin and seasonality of reproduction.

Wild and domesticated species display seasonality in reproductive function, controlled predominantly by photoperiod. Seasonal alterations in breeding status are caused by changes in the secretion of gonadotropin-releasing hormone (GnRH) that are mediated by upstream neuronal afferents that regulate the GnRH cells. In particular, kisspeptin appears to play a major role in seasonality of reproduction, transducing the feedback effect of gonadal steroids as well as having an independent (nonsteroid dependent) circannual rhythm. A substantial body of data on this issue has been obtained from studies in sheep and hamsters and this is reviewed here in detail. Kisspeptin function is upregulated during the breeding season in sheep, stimulating reproductive function, but contradictory data are found in Siberian and Syrian hamsters. The relative quiescence of kisspeptin cells in the nonbreeding season can be counteracted by administration of the peptide, leading to activation of reproductive function. Although there is a major role for melatonin in the transduction of photoperiod to the reproductive system, kisspeptin cells do not appear to express the melatonin receptor, so the means by which seasonality changes the level of kisspeptin activity remains unknown.

Gonadotropin-inhibitory hormone is a hypothalamic peptide that provides a molecular switch between reproduction and feeding.

OBJECTIVE: Gonadotropin-inhibitory hormone (GnIH)-3 is a neuropeptide that plays a major role in the regulation of reproduction and feeding in mammals. MATERIALS AND METHODS: We measured endocrine and behavioural parameters of reproduction in sheep, and sexual behaviour in sheep, mice and cynomolgus monkeys. In addition, GnIH gene expression (in situ hybridization) was examined in ewes, and effects of GnIH-3 on food intake and energy expenditure were measured in various species. GnIH-3 was infused (i.v.) into ewes after an i.m. injection of estradiol benzoate to determine whether the peptide blocks the surge in luteinizing hormone (LH) secretion. RESULTS: GnIH gene expression was reduced in the preovulatory period in ewes. Infusion (i.v.) of GnIH-3 blocked the estrogen-induced LH surge (in ewes). Intracerebroventricular infusion had no effect on female or male sexual behaviour in each of the three species, but increased food intake. There were no effects on energy expenditure in sheep or rats. GnIH increased fos protein (immunohistochemistry) was seen in orexigenic neurons (in sheep and rats), but also in anorexigenic neurons (in sheep). CONCLUSIONS: GnIH-3 reduces reproductive hormone levels and increases food intake in mammals without reducing energy expenditure. There is minimal effect on reproductive behaviour. The dual effect on reproduction and feeding suggests that GnIH-3 provides a molecular switch between these two functions. Blockade of the positive feedback effect of estrogen with parenteral infusion indicates that this peptide may have utility as a blocker of reproductive function in mammals.

The intimate relationship of gonadotropin-releasing hormone neurons with the polysialylated neural cell adhesion molecule revisited across development and adult plasticity.

The neurohormone gonadotropin-releasing hormone (GnRH) is critical for all the aspects of reproductive life in vertebrates. GnRH is secreted by a small number of neurons dispersed within the preoptic-hypothalamic region. These neurons are derived from the embryonic olfactory pit. They then migrate along olfactory, vomeronasal and terminal nerves to their final destination. Classical approaches to study the regulation of GnRH secretion during the reproductive cycle have focused on the various neuronal inputs on GnRH neurons and their regulation by ovarian steroids. However, it is well known that steroids will change the microenvironment of neuronal networks and can induce plasticity and functional changes. In this review, we will focus on the intimate relationship of developing and adult GnRH neurons with the polysialylated form of neural cell adhesion molecule (PSA-NCAM), a major molecular actor in the morphogenesis and adult plasticity of the nervous system. We will first recapitulate the spatiotemporal relationship between PSA-NCAM and migrating GnRH neurons during embryogenesis of various vertebrate species and discuss its importance for GnRH neuron development as shown by various loss of function studies. In the adult, we will review the relationships between PSA-NCAM and GnRH neurons across various physiological states, and open the discussion to the use of new model systems that can help to unravel the function and mechanism of action of PSA-NCAM on GnRH neuronal network activity and GnRH release.

Kisspeptin and seasonality in sheep.

Sheep are seasonal breeders, experiencing a period of reproductive quiescence during spring and early summer. During the non-breeding period, kisspeptin expression in the arcuate nucleus is markedly reduced. This strongly suggests that the mechanisms that control seasonal changes in reproductive function involve kisspeptin neurons. Kisspeptin cells appear to regulate GnRH neurons and transmit sex-steroid feedback to the reproductive axis. Since the non-breeding season is characterized by increased negative feedback of estrogen on GnRH secretion, the kisspeptin neurons seem to be fundamentally involved in the determination of breeding state. The reduction in kisspeptin neuronal function during the non-breeding season can be corrected by infusion of kisspeptin, which causes ovulation in seasonally acyclic females.

Identification and characterization of a new interleukin-8 receptor in bovine species.

Mastitis is an inflammation of the mammary gland, most of the time caused by invading pathogens. Phagocytosis by neutrophils is a crucial defense of the mammary gland and the prompt recruitment of these phagocytes from blood to milk compartments is essential for the outcome of the infection. ELR+ CXC chemokines, ligands of the two interleukin-8 receptors (IL-8R), CXCR1 and CXCR2, are likely to be involved in the initiation of the inflammatory response and also in the migration of neutrophils. Recently, the polymorphism of bovine CXCR2 has been associated with resistance to mastitis. However, as the bovine IL-8R are not functionally defined, their contribution to the recruitment of neutrophils remains undetermined. In this study, the RNA ligase-mediated (RLM)-RACE method was used to clone a novel bovine interleukin-8 receptor (nIL-8R) of the bovine species. We showed that both bovine IL-8R (nIL-8R and the published CXCR2) are functional since bovine IL-8 induced migration of HEK-293 cells expressing either IL-8R. In addition, comparisons of full-length sequences suggested that the published CXCR2 sequence was improperly annotated and that the sequences of the nIL-8R and the published CXCR2 are homologous to human CXCR2 and CXCR1, respectively. This was confirmed by binding assays with labeled IL-8 and GRO-beta and calcium (Ca) flux responses of transfected cells. Moreover, the C-terminal of both bovine IL-8R showed 100% identity, whereas they differ in most other species, suggesting that the two bovine IL-8R initiate similar signal transduction. These results constitute a basis to improve our understanding of the molecular mechanisms implicated in the recruitment of bovine neutrophils.

Gonadotropin-releasing hormone in third ventricular cerebrospinal fluid: endogenous distribution and exogenous uptake.

GnRH is detectable in the cerebrospinal fluid (CSF), but its source remains unidentified. Previous studies have harvested CSF for GnRH analysis from the median eminence region, but it is unknown whether GnRH in CSF is restricted to this region. If CSF-GnRH plays a physiological role, through volume transmission, to communicate with brain regions that express GnRH receptors but are not evidently innervated by GnRH neurons, then it is essential to establish whether GnRH is more pervasive throughout the cerebroventricular system. Three cannulae were placed in the supraoptic, infundibular, and pineal recesses of the third ventricle. GnRH was undetectable in lateral ventricle CSF. GnRH pulses were detected in all ewes in infundibular recess CSF, but at sites more rostral (supraoptic) and caudal (pineal), GnRH pulse frequency and amplitude significantly (P<0.05) decreased. A GnRH surge was evident in CSF collected simultaneously from all cannulae, but the amplitude was greatest (P<0.05) at the infundibular recess. A final study established whether iv administered GnRH enters the CSF. A 250-ng GnRH dose did not affect CSF-GnRH concentrations (1.6+/-0.3 pg/ml), but 2.5 microg (2.7+/-0.2 pg/ml; P<0.001) and 1 mg (38.5+/-10.6 pg/ml; P<0.05) significantly increased CSF-GnRH concentrations. The present study shows: 1) the median eminence region is likely to be the major, if not only, source of GnRH entering the cerebroventricular system; and 2) exogenous GnRH crosses the blood-brain barrier, but extremely high doses are required to elevate CSF concentrations to physiological levels. Thus, CSF-GnRH may affect sites that are closer in proximity to the infundibular recess region than previously thought.

KiSS-1 messenger ribonucleic acid expression in the hypothalamus of the ewe is regulated by sex steroids and season.

The KiSS-1 gene encodes a family of peptides called kisspeptins, which are endogenous ligands for the G protein-coupled receptor GPR54. Kisspeptin function appears to be critical for GnRH secretion and the initiation of puberty. To test the hypothesis that steroid hormones regulate KiSS-1 mRNA expression in the ewe, we examined the brains of ovary-intact (luteal phase) and ovariectomized (OVX) ewes, as well as OVX ewes that received estradiol (E) or progesterone (P) replacement. KiSS-1 mRNA-expressing cells were predominantly located in the arcuate nucleus (ARC). Here, expression was increased after OVX but returned to the level of gonad-intact animals with E treatment. Treatment with P partially restored KiSS-1 expression toward gonad-intact levels. Double-label immunohistochemistry revealed that approximately 86% of kisspeptin-immunoreactive cells in the ARC are also P-receptor positive. Finally, we tested the hypothesis that KiSS-1 mRNA is lower during anestrus, due to a non-steroid-dependent seasonal effect. In OVX ewes, expression in the ARC was lower at the time of year corresponding to anestrus. We conclude that KiSS-1 expression in the ARC of the ewe brain is negatively regulated by chronic levels of E and P, suggesting that both steroids may exert negative feedback control on GnRH secretion through altered kisspeptin signaling. Furthermore, a seasonal alteration in KiSS-1 expression in the ARC of OVX ewes strongly suggests that kisspeptin is fundamentally involved in the control of seasonal changes in reproductive function.

Kisspeptin neurons in the arcuate nucleus of the ewe express both dynorphin A and neurokinin B.

Kisspeptin is a potent stimulator of GnRH secretion that has been implicated in the feedback actions of ovarian steroids. In ewes, the majority of hypothalamic kisspeptin neurons are found in the arcuate nucleus (ARC), with a smaller population located in the preoptic area. Most arcuate kisspeptin neurons express estrogen receptor-alpha, as do a set of arcuate neurons that contain both dynorphin and neurokinin B (NKB), suggesting that all three neuropeptides are colocalized in the same cells. In this study we tested this hypothesis using dual immunocytochemistry and also determined if kisspeptin neurons contain MSH or agouti-related peptide. To assess colocalization of kisspeptin and dynorphin, we used paraformaldehyde-fixed tissue from estrogen-treated ovariectomized ewes in the breeding season (n = 5). Almost all ARC, but no preoptic area, kisspeptin neurons contained dynorphin. Similarly, almost all ARC dynorphin neurons contained kisspeptin. In experiment 2 we examined colocalization of kisspeptin and NKB in picric-acid fixed tissue collected from ovary intact ewes (n = 9). Over three quarters of ARC kisspeptin neurons also expressed NKB, and a similar percentage of NKB neurons contained kisspeptin. In contrast, no kisspeptin neurons stained for MSH or agouti-related peptide. These data demonstrate that, in the ewe, a high percentage of ARC kisspeptin neurons also produce dynorphin and NKB, and we propose that a single subpopulation of ARC neurons contains all three neuropeptides. Because virtually all of these neurons express estrogen and progesterone re-ceptors, they are likely to relay the feedback effects of these steroids to GnRH neurons to regulate reproductive function.

Molecular mechanisms involved in LH release by the ovine pituitary cells.

The luteinizing hormone-releasing hormone (LHRH) is a hypothalamic decapeptide and main positive regulator of luteinizing hormone (LH) secretion from pituitary cells. Insulin-like growth factor-I (IGF-1) also stimulates LH release and enhances the effect of LHRH. However, the molecular mechanisms involved in the interactions between LHRH and IGF-1 are unclear. Here, we first determined the effect of various types of LHRH [I (mammalian), II (chicken), III (lamprey), hyp9 and salmon] on both LH secretion and activation of MAPK (ERK1/2 and p38) in ovine pituitary cells. After 3h of treatment, LH secretion was significantly higher for LHRH-I than for the other LHRH tested. Interestingly, LHRH-III had no effect at any concentration used on the LH release by ovine pituitary cells. The phosphorylation of both MAPK ERK1/2 and p38 was also significantly higher after treatment with LHRH-I than LHRH-II, salmon LHRH or hyp9. These MAPKs were not activated or only very weakly activated by LHRH-III. We then used pharmacological inhibitors to show that MAPK ERK1/2 and PKCdelta participate in the LH release by ovine pituitary cells in response to LHRH-I. We identified the main substrates and signaling pathways [PI3K/Akt and MAPK (ERK1/2, p38 and JNK1/2] of IGF-1R and investigated the effect of IGF-1 on the stimulation of ovine pituitary cell LH secretion by the various LHRH. IGF-1 increases LH secretion in response to LHRH-I, LHRH-II, hyp9 and salmon LHRH but not the secretion after treatment with LHRH-III. Using specific inhibitors, we found that the MAPK ERK1/2 but not the PI3K/Akt signaling pathway is involved in the LH secretion in response to IGF-1. This is the first description of a common molecular mechanism, involving the MAPK ERK1/2, by which LHRH-R and IGF-1-R induce LH secretion in ovine pituitary cells.

Rational design of triazololipopeptides analogs of kisspeptin inducing a long-lasting increase of gonadotropins.

New potent and selective KISS1R agonists were designed using a combination of rational chemical modifications of the endogenous neuropeptide kisspeptin 10 (KP10). Improved resistance to degradation and presumably reduced renal clearance were obtained by introducing a 1,4-disubstituted 1,2,3-triazole as a proteolysis-resistant amide mimic and a serum albumin-binding motif, respectively. These triazololipopeptides are highly potent full agonists of KISS1R and are >100 selective over the closely related NPFF1R. When injected in ewes with a quiescent reproductive system, the best compound of our series induced a much prolonged increase of luteinizing hormone release compared to KP10 and increased follicle-stimulating hormone plasma concentration. Hence, this KISS1R agonist is a new valuable pharmacological tool to explore the potential of KP system in reproduction control. Furthermore, it represents the first step to develop drugs treating reproductive system disorders due to a reduced activity of the hypothalamo-pituitary-gonadal axis such as delayed puberty, hypothalamic amenorrhea, and hypogonadotropic hypogonadism.

Neuroanatomical organization of gonadotropin-releasing hormone neurons during the oestrus cycle in the ewe.

BACKGROUND: During the preovulatory surge of gonadotropin-releasing hormone (GnRH), a very large amount of the peptide is released in the hypothalamo-hypophyseal portal blood for 24-36H00. To study whether this release is linked to a modification of the morphological organization of the GnRH-containing neurons, i.e. morphological plasticity, we conducted experiments in intact ewes at 4 different times of the oestrous cycle (before the expected LH surge, during the LH surge, and on day 8 and day 15 of the subsequent luteal phase). The cycle stage was verified by determination of progesterone and LH concentrations in the peripheral blood samples collected prior to euthanasia. RESULTS: The distribution of GnRH-containing neurons throughout the preoptic area around the vascular organ of the lamina terminalis was studied following visualisation using immunohistochemistry. No difference was observed in the staining intensity for GnRH between the different groups. Clusters of GnRH-containing neurons (defined as 2 or more neurons being observed in close contact) were more numerous during the late follicular phase (43 +/- 7) than during the luteal phase (25 +/- 6), and the percentage of clusters was higher during the beginning of the follicular phase than during the luteal phase. There was no difference in the number of labelled neurons in each group. CONCLUSIONS: These results indicate that the morphological organization of the GnRH-containing neurons in ewes is modified during the follicular phase. This transitory re-organization may contribute to the putative synchronization of these neurons during the surge. The molecular signal inducing this plasticity has not yet been identified, but oestradiol might play an important role, since in sheep it is the only signal which initiates the GnRH preovulatory surge.

Cellular mechanisms and integrative timing of neuroendocrine control of GnRH secretion by kisspeptin.

The hypothalamus integrates endogenous and exogenous inputs to control the pituitary-gonadal axis. The ultimate hypothalamic influence on reproductive activity is mediated through timely secretion of GnRH in the portal blood, which modulates the release of gonadotropins from the pituitary. In this context neurons expressing the RF-amide neuropeptide kisspeptin present required features to fulfill the role of the long sought-after hypothalamic integrative centre governing the stimulation of GnRH neurons. Here we focus on the intracellular signaling pathways triggered by kisspeptin through its cognate receptor KISS1R and on the potential role of proteins interacting with this receptor. We then review evidence implicating both kisspeptin and RFRP3--another RF-amide neuropeptide--in the temporal orchestration of both the pre-ovulatory LH surge in female rodents and the organization of seasonal breeding in photoperiodic species.

Seasonal changes in RFamide-related peptide-3 neurons in the hypothalamus of a seasonally breeding marsupial species, the brushtail possum (Trichosurus vulpecula).

RFamide-related peptide-3 (RFRP-3) neurons have been shown to inhibit gonadotropin-releasing hormone (GnRH) neuronal activity and hence reproduction in birds and eutherian mammals. They have also been proposed to have a direct hypophysiotropic effect on pituitary gonadotropin release. We used a new RFRP-3 antibody to characterize the cell body distribution and fiber projections of RFRP-3 neurons in the adult female brushtail possum brain. RFRP-3-immunoreactive cell bodies were found scattered within the dorsomedial hypothalamus and the dorsomedial half of the ventromedial hypothalamus, while GnRH neurons were observed scattered rostrocaudally along the lateral septum, rostral to the medial septum. There was a significant 2-fold increase in the RFRP-3 cell body number during the nonbreeding season (summer) compared to the breeding season (winter). Immunoreactive RFRP-3 fibers were distributed throughout the thalamus, preoptic area, and hypothalamus. Very few fibers were observed in the median eminence, especially in the external zone. Intraperitoneal injection of the retrograde tracer Fluoro-Gold resulted in the labeling of 40% of hypophysiotropic tuberoinfundibular dopaminergic (tyrosine hydroxylase-positive) neurons; however, <10% of zona incerta dopaminergic neurons (which are not hypophysiotropic) or RFRP-3 neurons were labeled with this tracer. These observations suggest that RFRP-3 exhibits a seasonal fluctuation in cell numbers, as seen in sheep and birds, which is consistent with an increased inhibitory tone during the nonbreeding season. The lack of RFRP-3 fibers in the median eminence and of Fluoro-Gold uptake from the periphery imply that the actions of this peptide occur primarily centrally rather than at the anterior pituitary gland.

Socially regulated reproductive development: analysis of GnRH-1 and kisspeptin neuronal systems in cooperatively breeding naked mole-rats (Heterocephalus glaber).

In naked mole-rat (NMR) colonies, breeding is monopolized by the queen and her consorts. Subordinates experience gonadal development if separated from the queen. To elucidate the neuroendocrine factors underlying reproductive suppression/development in NMRs, we quantified plasma gonadal steroids and GnRH-1- and kisspeptin-immunoreactive (ir) neurons in subordinate adults and in those allowed to develop into breeders, with or without subsequent gonadectomy. In males and females, respectively, plasma testosterone and progesterone are higher in breeders than in subordinates. No such distinction occurs for plasma estradiol; its presence after gonadectomy and its positive correlation with adrenal estradiol suggest an adrenal source. Numbers of GnRH-1-ir cell bodies do not differ between gonad-intact breeders and subordinates within or between the sexes. As in phylogenetically related guinea pigs, kisspeptin-ir processes pervade the internal and external zones of the median eminence. Their distribution is consistent with actions on GnRH-1 neurons at perikaryal and/or terminal levels. In previously investigated species, numbers of kisspeptin-ir cell bodies vary from substantial to negligible according to sex and/or reproductive state. NMRs are exceptional: irrespective of sex, reproductive state, or presence of gonads, substantial numbers of kisspeptin-ir cell bodies are detected in the rostral periventricular region of the third ventricle (RP3V) and in the anterior periventricular (PVa), arcuate, and dorsomedial hypothalamic nuclei. Nevertheless, the greater number in the RP3V/PVa of female breeders compared with female subordinates or male breeders suggests that emergence from a hypogonadotrophic state in females may involve kisspeptin-related mechanisms similar to those underlying puberty or seasonal breeding in other species.

Redundancy in Kiss1 expression safeguards reproduction in the mouse.

Kisspeptin (Kiss1) signaling to GnRH neurons is widely acknowledged to be a prerequisite for puberty and reproduction. Animals lacking functional genes for either kisspeptin or its receptor exhibit low gonadotropin secretion and infertility. Paradoxically, a recent study reported that genetic ablation of nearly all Kiss1-expressing neurons (Kiss1 neurons) does not impair reproduction, arguing that neither Kiss1 neurons nor their products are essential for sexual maturation. We posited that only minute quantities of kisspeptin are sufficient to support reproduction. If this were the case, animals having dramatically reduced Kiss1 expression might retain fertility, testifying to the redundancy of Kiss1 neurons and their products. To test this hypothesis and to determine whether males and females differ in the required amount of kisspeptin needed for reproduction, we used a mouse (Kiss1-CreGFP) that has a severe reduction in Kiss1 expression. Mice that are heterozygous and homozygous for this allele (Kiss1(Cre/+) and Kiss1(Cre/Cre)) have ∼50% and 95% reductions in Kiss1 transcript, respectively. We found that although male Kiss1(Cre/Cre) mice sire normal-sized litters, female Kiss1(Cre/Cre) mice exhibit significantly impaired fertility and ovulation. These observations suggest that males require only 5% of normal Kiss1 expression to be reproductively competent, whereas females require higher levels for reproductive success.