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.     

Leptin Responsive and GABAergic Projections to the Rostral Preoptic Area in Mice

The adipocyte‐derived hormone leptin plays a critical role in the control of reproduction via signalling in hypothalamic neurones. The drivers of the hypothalamic‐pituitary‐gonadal axis, the gonadotrophin‐releasing hormone (GnRH) neurones, do not have the receptors for leptin. Therefore, intermediate leptin responsive neurones must provide leptin‐to‐GnRH signalling. We investigated the populations of leptin responsive neurones that provide input to the rostral preoptic area (rPOA) where GnRH cell bodies reside. Fluorescent retrograde tracer beads (RetroBeads; Lumafluor Inc., Naples, FL, USA) were injected into the rPOA of transgenic leptin receptor enhanced green fluorescent protein (Lepr‐eGFP) reporter mice. Uptake of the RetroBeads by Lepr‐eGFP neurones was assessed throughout the hypothalamus. RetroBead uptake was most evident in the medial arcuate nucleus (ARC), the dorsomedial nucleus (DMN) and the ventral premammillary nucleus (PMV) of the hypothalamus. The uptake of RetroBeads specifically by Lepr‐eGFP neurones was highest in the medial ARC (18% of tracer‐labelled neurones Lepr‐eGFP‐positive). Because neurones that are both leptin responsive and GABAergic play a critical role in the regulation of fertility by leptin, we next focussed on the location of these populations. To address whether GABAergic neurones in leptin‐responsive hypothalamic regions project to the rPOA, the experiment was repeated in GABA neurone reporter mice (Vgat‐tdTomato). Between 10% and 45% of RetroBead‐labelled neurones in the ARC were GABAergic, whereas uptake of tracer by GABAergic neurones in the DMN and PMV was very low (< 5%). These results show that both leptin responsive and GABAergic neurones from the ARC project to the region of the GnRH cell bodies. Our findings suggest that LEPR‐expressing GABA neurones from the ARC may be mediators of leptin‐to‐GnRH signalling.     

Kisspeptin Neurones do not Directly Signal to RFRP‐3 Neurones but RFRP‐3 may Directly Modulate a Subset of Hypothalamic Kisspeptin Cells in Mice

The neuropeptides kisspeptin (encoded by Kiss1) and RFamide‐related peptide‐3 (also known as GnIH; encoded by Rfrp) are potent stimulators and inhibitors, respectively, of reproduction. Whether kisspeptin or RFRP‐3 might act directly on each other's neuronal populations to indirectly modulate reproductive status is unknown. To examine possible interconnectivity of the kisspeptin and RFRP‐3 systems, we performed double‐label in situ hybridisation (ISH) for the RFRP‐3 receptors, Gpr147 and Gpr74, in hypothalamic Kiss1 neurones of adult male and female mice, as well as double‐label ISH for the kisspeptin receptor, Kiss1r, in Rfrp‐expressing neurones of the hypothalamic dorsal‐medial nucleus (DMN). Only a very small proportion (5‐10%) of Kiss1 neurones of the anteroventral periventricular region expressed Gpr147 or Gpr74 in either sex, whereas higher co‐expression (approximately 25%) existed in Kiss1 neurones in the arcuate nucleus. Thus, RFRP‐3 could signal to a small, primarily arcuate, subset of Kiss1 neurones, a conclusion supported by the finding of approximately 35% of arcuate kisspeptin cells receiving RFRP‐3‐immunoreactive fibre contacts. By contrast to the former situation, no Rfrp neurones co‐expressed Kiss1r in either sex, and Tacr3, the receptor for neurokinin B (NKB; a neuropeptide co‐expressed with arcuate kisspeptin neurones) was found in <10% of Rfrp neurones. Moreover, kisspeptin‐immunoreactive fibres did not readily appose RFRP‐3 cells in either sex, further excluding the likelihood that kisspeptin neurones directly communicate to RFRP‐3 neurones. Lastly, despite abundant NKB in the DMN region where RFRP‐3 soma reside, NKB was not co‐expressed in the majority of Rfrp neurones. Our results suggest that RFRP‐3 may modulate a small proportion of kisspeptin‐producing neurones in mice, particularly in the arcuate nucleus, whereas kisspeptin neurones are unlikely to have any direct reciprocal actions on RFRP‐3 neurones.     

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

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

Isolation and identification of endogenous RFamide‐related peptides 1 and 3 in the mouse hypothalamus

Although the RFamide‐related peptide (RFRP) preproprotein sequence is known in mice, until now, the molecular structure of the mature, functional peptides processed from the target precursor molecule has not been determined. In the present study, we purified endogenous RFRP1 and RFRP3 peptides from mouse hypothalamic tissue extracts using an immunoaffinity column conjugated with specific antibodies against the mouse C‐terminus of RFRP‐1 and RFRP‐3. Employing liquid chromatography coupled with mass spectrometry, we demonstrated that RFRP1 consists of 15 amino acid residues and RFRP3 consists of 10 amino acid residues (ANKVPHSAANLPLRF‐NH2 and SHFPSLPQRF‐NH2, respectively). To investigate the distribution of RFRPs in the mouse central nervous system, we performed immunohistochemical staining of the brain sections collected from wild‐type and Rfrp knockout animals. These data, together with gene expression in multiple tissues, provide strong confidence that RFRP‐immunoreactive neuronal cells are localised in the dorsomedial hypothalamic nucleus (DMH) and between the DMH and the ventromedial hypothalamic nuclei. The identification of RFRP1 and RFRP3 peptides and immunohistochemical visualisation of targeting RFRPs neurones in the mice brain provide the basis for further investigations of the functional biology of RFRPs.     

Leptin increases striatal dopamine D2 receptor binding in leptin‐deficient obese (ob/ob) mice

Peripheral and central leptin administration have been shown to mediate central dopamine (DA) signaling. Leptin‐receptor deficient rodents show decreased DA D2 receptor (D2R) binding in striatum and unique DA profiles compared to controls. Leptin‐deficient mice show increased DA activity in reward‐related brain regions. The objective of this study was to examine whether basal D2R‐binding differences contribute to the phenotypic behaviors of leptin‐deficient ob/ob mice, and whether D2R binding is altered in response to peripheral leptin treatment in these mice. Leptin decreased body weight, food intake, and plasma insulin concentration in ob/ob mice but not in wild‐type mice. Basal striatal D2R binding (measured with autoradiography [³H] spiperone) did not differ between ob/ob and wild‐type mice but the response to leptin did. In wild‐type mice, leptin decreased striatal D2R binding, whereas, in ob/ob mice, leptin increased D2R binding. Our findings provide further evidence that leptin modulates D2R expression in striatum and that these effects are genotype/phenotype dependent. Synapse 64:503–510, 2010. © 2010 Wiley‐Liss, Inc.     

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

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

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

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

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

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

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. J. Comp. Neurol. 521: 3030–3041, 2013. © 2013 Wiley Periodicals, Inc.     

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

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

Evidence 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.     

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

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

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

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

Distribution and Seasonal Variation in Hypothalamic RF‐amide Peptides in a Semi‐Desert Rodent,the Jerboa

The jerboa is a semi‐desert rodent, in which reproductive activity depends on the seasons, being sexually active in the spring–summer. The present study aimed to determine whether the expression of two RF‐amide peptides recently described to regulate gonadotrophin‐releasing hormone neurone activity, kisspeptin (Kp) and RF‐amide‐related peptide (RFRP)‐3, displays seasonal variation in jerboa. Kp and/or RFRP‐3 immunoreactivity was investigated in the hypothalamus of jerboas captured in the field of the Middle Atlas mountain (Morocco), either in the spring or autumn. As in other rodents, the Kp‐immunoreactive (‐IR) neurones were found in the anteroventro‐periventricular and arcuate nuclei. RFRP‐3 neurones were noted within the dorso/ventromedial hypothalamus. A marked sexual dimorphism in the expression of Kp (but not RFRP‐3) was observed. The number of Kp‐IR neurones was nine‐fold higher, and the density of Kp‐IR fibres and terminal‐like elements in the median eminence was two‐fold higher in females than in males. Furthermore, a significant seasonal variation in peptide expression was obtained with an increase in both Kp‐ and RFRP‐3‐IR cell bodies in sexually active male jerboas captured in the spring compared to sexually inactive autumn animals. In the arcuate nucleus, the level of Kp‐IR cells and fibres was significant higher during the sexually active period in the spring than during the autumnal sexual quiescence. Similarly, the number of RFRP‐3‐IR neurones in the ventro/dorsomedial hypothalamus was approximately three‐fold higher in sexually active jerboa captured in the spring compared to sexually inactive autumn animals. Altogether, the present study reports the distribution of Kp and RFRP‐3 neurones in the hypothalamus of a desert species and reveals a seasonal difference in their expression that correlates with sexual activity. These findings suggest that these two RF‐amide peptides may act in concert to synchronise the gonadotrophic activity of jerboas with the seasons.     

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.