Prenatal Testosterone Excess Decreases Neurokinin 3 Receptor Immunoreactivity within the Arcuate Nucleus KNDy Cell Population

Prenatal exposure of the female ovine foetus to excess testosterone leads to neuroendocrine disruptions in adulthood, as demonstrated by defects in responsiveness with respect to the ability of gonadal steroids to regulate gonadotrophin‐releasing hormone (GnRH) secretion. In the ewe, neurones of the arcuate nucleus (ARC), which co‐expresses kisspeptin, neurokinin B (NKB) and dynorphin (termed KNDy cells), play a key role in steroid feedback control of GnRH and show altered peptide expression after prenatal testosterone treatment. KNDy cells also co‐localise NKB receptors (NK3R), and it has been proposed that NKB may act as an autoregulatory transmitter in KNDy cells where it participates in the mechanisms underlying steroid negative‐feedback. In addition, recent evidence suggests that NKB/NK3R signalling may be involved in the positive‐feedback actions of oestradiol leading to the GnRH/luteinising hormone (LH) surge in the ewe. Thus, we hypothesise that decreased expression of NK3R in KNDy cells may be present in the brains of prenatal testosterone‐treated animals, potentially contributing to reproductive defects. Using single‐ and dual‐label immunohistochemistry we found NK3R‐positive cells in diverse areas of the hypothalamus; however, after prenatal testosterone treatment, decreased numbers of NK3R immunoreactive (‐IR) cells were seen only in the ARC. Moreover, dual‐label confocal analyses revealed a significant decrease in the percentage of KNDy cells (using kisspeptin as a marker) that co‐localised NK3R. To investigate how NKB ultimately affects GnRH secretion in the ewe, we examined GnRH neurones in the preoptic area (POA) and mediobasal hypothalamus (MBH) for the presence of NK3R. Although, consistent with earlier findings, we found no instances of NK3R co‐localisation in GnRH neurones in either the POA or MBH; in addition, > 70% GnRH neurones in both areas were contacted by NK3R‐IR presynaptic terminals suggesting that, in addition to its role at KNDy cell bodies, NKB may regulate GnRH neurones by presynaptic actions. In summary, the finding of decreased NK3R within KNDy cells in prenatal testosterone‐treated sheep complements previous observations of decreased NKB and dynorphin in the same population, and may contribute to deficits in the feedback control of GnRH/LH secretion in this animal model.     

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.     

Colocalisation of dynorphin a and neurokinin B immunoreactivity in the arcuate nucleus and median eminence of the sheep

Dynorphin A (DYN)-containing cells play a key role in conveying the negative feedback influence of progesterone upon pulsatile gonadotrophin-releasing hormone (GnRH) secretion in the ewe. A very high percentage of DYN cells in the arcuate nucleus express the progesterone receptor; another population of arcuate nucleus cells that also express steroid receptors in the sheep are those that express the tachykinin peptide, neurokinin B (NKB). Both DYN and NKB fibres have been shown to form close contacts with ovine GnRH cells. Therefore, the present study tested the hypothesis that neurones expressing NKB and DYN represent the same neuronal population in the arcuate nucleus. Confocal microscopic analysis of brain sections processed for dual immunofluorescence revealed that a large majority of DYN neurones in the arcuate nucleus were also immunoreactive for NKB. Likewise, a similar majority of NKB neurones in the arcuate nucleus were immunoreactive for DYN. By contrast, DYN cells in the preoptic area and anterior hypothalamus did not colocalise with NKB, nor did DYN cells in the paraventricular or supraoptic nuclei. Fibres that stained positively for both DYN and NKB were seen in the arcuate nucleus, where they formed close appositions with DYN/NKB-positive neurones, and in the external zone of the median eminence. Taken together with previous findings, these data suggest that a subpopulation of arcuate nucleus neurones coexpressing DYN and NKB mediate the negative feedback influence of progesterone on pulsatile GnRH secretion in the ewe and may also be involved in other feedback actions of gonadal steroids.     

Evidence for Changes in Numbers of Synaptic Inputs onto KNDy and GnRH Neurones during the Preovulatory LH Surge in the Ewe

Kisspeptin neurones located in the arcuate nucleus (ARC) and preoptic area (POA) are critical mediators of gonadal steroid feedback onto gonadotrophin‐releasing hormone (GnRH) neurones. ARC kisspeptin cells that co‐localise neurokinin B (NKB) and dynorphin (Dyn), are collectively referred to as KNDy (Kisspeptin/NKB/Dyn) neurones, and have been shown in mice to also co‐express the vesicular glutamate transporter, vGlut2, an established glutamatergic marker. The ARC in rodents has long been known as a site of hormone‐induced neuroplasticity, and changes in synaptic inputs to ARC neurones in rodents occur over the oestrous cycle. Based on this evidence, the the present study aimed to examine possible changes across the ovine oestrous cycle in synaptic inputs onto kisspeptin cells in the ARC (KNDy) and POA, and inputs onto GnRH neurones. Gonadal‐intact breeding season ewes were perfused using 4% paraformaldehyde during either the luteal or follicular phase of the oestrous cycle, with the latter group killed at the time of the luteinising hormone (LH) surge. Hypothalamic sections were processed for triple‐label immunodetection of kisspeptin/vGlut2/synaptophysin or kisspeptin/vGlut2/GnRH. The total numbers of synaptophysin‐ and vGlut2‐positive inputs to ARC KNDy neurones were significantly increased at the time of the LH surge compared to the luteal phase; because these did not contain kisspeptin, they do not arise from KNDy neurones. By contrast to the ARC, the total number of synaptophysin‐positive inputs onto POA kisspeptin neurones did not differ between luteal phase and surge animals. The total number of kisspeptin and vGlut2 inputs onto GnRH neurones in the mediobasal hypothalamus (MBH) was also increased during the LH surge, and could be attributed to an increase in the number of KNDy (double‐labelled kisspeptin + vGlut2) inputs. Taken together, these results provide novel evidence of synaptic plasticity at the level of inputs onto KNDy and GnRH neurones during the ovine oestrous cycle. Such changes may contribute to the generation of the preovulatory GnRH/LH surge.     

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.     

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

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

Kisspeptin,gonadotrophin‐releasing hormone and oestrogen receptor α colocalise with neuronal nitric oxide synthase neurones in prepubertal female sheep

Puberty is a process that integrates multiple inputs ultimately resulting in an increase in gonadotrophin‐releasing hormone (GnRH) secretion. Although kisspeptin neurones play an integral role in GnRH secretion and puberty onset, other systems are also likely important. One potential component is nitric oxide (NO), a gaseous neurotransmitter synthesised by nitric oxide synthase (NOS). The present study aimed to neuroanatomically characterise neuronal NOS (nNOS) in prepubertal female sheep and determine whether oestradiol exerts effects on this system. Luteinising hormone secretion was reduced by oestradiol treatment in prepubertal ovariectomised ewes. Neurones immunoreactive for nNOS were identified in several areas, with the greatest number present in the ventrolateral portion of the ventromedial hypothalamus, followed by the ventromedial hypothalamus, preoptic area (POA) and arcuate nucleus (ARC). Next, we determined whether nNOS neurones contained oestrogen receptor (ER)α and could potentially communicate oestradiol (E₂) feedback to GnRH neurones. Neuronal NOS neurones contained ERα with the percentage of coexpression (12%‐40%) depending upon the area analysed. We next investigated whether a neuroanatomical relationship existed between nNOS and kisspeptin or nNOS and GnRH neurones. A high percentage of kisspeptin neurones in the POA (79%) and ARC (98%) colocalised with nNOS. Kisspeptin close contacts were also associated with nNOS neurones. A greater number of close contacts were observed in the ARC than the POA. A high percentage of POA GnRH neurones (79%) also expressed nNOS, although no GnRH close contacts were observed onto nNOS neurones. Neither the numbers of nNOS neurones in the POA or hypothalamus, nor the percentage of nNOS coexpression with GnRH, kisspeptin or ERα were influenced by oestradiol. These experiments reveal that a neuroanatomical relationship exists between both nNOS and kisspeptin and nNOS and GnRH in prepubertal ewes. Therefore, nNOS may act both directly and indirectly to influence GnRH secretion in prepubertal sheep.     

Neurokinin‐3 Receptor Activation in the Retrochiasmatic Area is Essential for the Full Pre‐Ovulatory Luteinising Hormone Surge in Ewes

Neurokinin B (NKB) is essential for human reproduction and has been shown to stimulate luteinising hormone (LH) secretion in several species, including sheep. Ewes express the neurokinin‐3 receptor (NK3R) in the retrochiasmatic area (RCh) and there is one report that placement of senktide, an NK3R agonist, therein stimulates LH secretion that resembles an LH surge in ewes. In the present study, we first confirmed that local administration of senktide to the RCh produced a surge‐like increase in LH secretion, and then tested the effects of this agonist in two other areas implicated in the control of LH secretion and where NK3R is found in high abundance: the preoptic area (POA) and arcuate nucleus (ARC). Bilateral microimplants containing senktide induced a dramatic surge‐like increase in LH when given in the POA similar to that seen with RCh treatment. By contrast, senktide treatment in the ARC resulted in a much smaller but significant increase in LH concentrations suggestive of an effect on tonic secretion. The possible role of POA and RCh NK3R activation in the LH surge was next tested by treating ewes with SB222200, an NK3R antagonist, in each area during an oestradiol‐induced LH surge. SB222200 in the RCh, but not in the POA, reduced the LH surge amplitude by approximately 40% compared to controls, indicating that NK3R activation in the former region is essential for full expression of the pre‐ovulatory LH surge. Based on these data, we propose that the actions of NKB in the RCh are an important component of the pre‐ovulatory LH surge in ewes.     

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.     

Neurokinin B Signalling in Human Puberty

Recent identification of TAC3 or TACR3 (encoding neurokinin B and its receptor, NK3R, respectively) mutations as the causes of normosmic idiopathic hypogonadotrophic hypogonadism has provided compelling evidence for the involvement of neurokinin B (NKB) signalling in puberty. A surge of subsequent studies pointing towards an understanding of the exact mechanism through which NKB signalling exerts its effects in puberty led to a postulated sketch of the GnRH pulse generator, in which kisspeptin, NKB and dynorphin work in concert to elicit pulsatile gonadotrophin‐releasing hormone release in the median eminence.     

Coexpression of dynorphin and neurokinin B immunoreactivity in the rat hypothalamus: Morphologic evidence of interrelated function within the arcuate nucleus

Considerable evidence suggests that dynorphin and neurokinin B (NKB) neurons in the hypothalamic arcuate nucleus participate in the sex-steroid regulation of reproduction. In the present study, we used dual-label immunofluorescence to explore the distribution of prodynorphin and proNKB immunoreactivity in the rat hypothalamus. Additionally, we investigated whether arcuate prodynorphin-ir (immunoreactive) neurons expressed the neurokinin 3 receptor (NK3R) or nuclear estrogen receptor-alpha (ERalpha). We found that the majority of prodynorphin-ir neurons in the rat arcuate nucleus expressed proNKB, whereas nearly all (99%) of the proNKB neurons were immunoreactive for prodynorphin. The arcuate nucleus was the only site in the hypothalamus where neuronal somata coexpressing prodynorphin and proNKB-immunoreactivity were identified. A dense plexus of double-labeled prodynorphin/proNKB-ir fibers was found within the arcuate nucleus extending to the median eminence and throughout the periventricular zone of the hypothalamus. Prodynorphin/proNKB fibers were also identified in the paraventricular nucleus, anterior hypothalamic area, medial preoptic area, median preoptic nucleus, anteroventral periventricular nucleus, and bed nucleus of the stria terminalis in a distribution consistent with previously described arcuate nucleus projections. Interestingly, the majority of prodynorphin-ir neurons in the arcuate nucleus expressed NK3R, and nearly 100% of the prodynorphin-ir neurons contained nuclear ERalpha. Our results suggest that there is a close functional relationship between dynorphin and NKB peptides within the arcuate nucleus of the rat, which may include an autofeedback loop mediated through NK3R. The diverse hypothalamic projections of fibers expressing both prodynorphin and proNKB provide evidence that these neurons may participate in a variety of homeostatic and neuroendocrine processes.     

Developmental changes in hypothalamic Kiss1 expression during activation of the pulsatile release of luteinising hormone in maturing ewe lambs

Onset of puberty is characterised by a marked increase in the frequency of release of gonadotrophin-releasing hormone (GnRH) and luteinising hormone (LH). The Kiss1 gene plays a critical role in pubertal development, and its product, kisspeptin, stimulates GnRH and LH release. In the present study, we tested the hypothesis that Kiss1 gene expression in the preoptic area (POA) and hypothalamus increases during maturation of the reproductive neuroendocrine axis in association with increased LH pulsatility. Ovariectomised, oestradiol-replaced lambs were euthanised at 25, 30 and 35 weeks of age. Blood samples were collected before euthanasia to characterise the pattern of LH release. Kiss1 mRNA was detected in coronal sections of the POA and hypothalamus and Kiss1-expressing cells were identified on the basis of silver grain density. The mean number of Kiss1-expressing cells in the POA/periventricular (PeV) areas increased from 25 to 30 weeks of age. No further increase at 35 weeks of age was observed, and the changes in Kiss1 expression in the POA/PeV were independent of changes in LH pulse frequency. The mean number of Kiss1-expressing cells in the arcuate (ARC) nucleus did not differ among age groups, although it was greater in the middle ARC of lambs exhibiting increased frequency of LH release. The density of silver grains per cell did not differ among groups in any of the areas studied. The results obtained indicate that the Kiss1 gene is activated in the POA/PeV and ARC of ewe lambs during juvenile development, and that kisspeptin neurones in the middle ARC, in particular, are involved in the acceleration of pulsatile LH release during maturation of the reproductive neuroendocrine axis in ewe lambs.     

Morphological and functional evidence for sexual dimorphism in neurokinin B signalling in the retrochiasmatic area of sheep

Neurokinin B (NKB) is critical for fertility in humans and stimulates gonadotrophin‐releasing hormone/luteinising hormone (LH) secretion in several species, including sheep. There is increasing evidence that the actions of NKB in the retrochiasmatic area (RCh) contribute to the induction of the preovulatory LH surge in sheep. In the present study, we determined whether there are sex differences in the response to RCh administration of senktide, an agonist to the NKB receptor (neurokinin receptor‐3 [NK3R]), and in NKB and NK3R expression in the RCh of sheep. To normalise endogenous hormone concentrations, animals were gonadectomised and given implants to mimic the pattern of ovarian steroids seen in the oestrous cycle. In females, senktide microimplants in the RCh produced an increase in LH concentrations that lasted for at least 8 hours after the start of treatment, whereas a much shorter increment (approximately 2 hours) was seen in males. We next collected tissue from gonadectomised lambs 18 hours after the insertion of oestradiol implants that produce an LH surge in female, but not male, sheep for immunohistochemical analysis of NKB and NK3R expression. As expected, there were more NKB‐containing neurones in the arcuate nucleus of females than males. Interestingly, there was a similar sexual dimorphism in NK3R‐containing neurones in the RCh, NKB‐containing close contacts onto these RCh NK3R neurones, and overall NKB‐positive fibres in this region. These data demonstrate that there are both functional and morphological sex differences in NKB‐NK3R signalling in the RCh and raise the possibility that this dimorphism contributes to the sex‐dependent ability of oestradiol to induce an LH surge in female sheep.     

The Effects of Neurokinin B upon Gonadotrophin Release in Male Rodents

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

Evidence in favour of a direct input from the ventromedial nucleus to gonadotropin-releasing hormone neurones in the ewe: an anterograde tracing study

The mechanism by which oestrogen activates the gonadotropin releasing hormone (GnRH) neurones to induce the preovulatory luteinizing hormone (LH) surge is not understood. Previous work in the ewe has suggested that the primary site of action for oestradiol in stimulating the GnRH neurones was in the region of the ventromedial nucleus (VMN) within the mediobasal hypothalamus (MBH). In the present study, we used anterograde tracing techniques in the ewe to investigate whether direct neuronal projections may exist from neurones located in the region of the VMN to the GnRH neurones. Following the injection of biotinylated dextran amine into the VMN of four ewes, anterogradely labelled fibres were found located principally within the ipsilateral diagonal band of Broca (DBB), septum, preoptic and anterior hypothalamic areas, and periventricular, paraventricular, dorsomedial and arcuate nuclei of the MBH. Dual-labelling for GnRH revealed that fibres containing anterograde tracer were adjacent to the soma and/or dendrites of approximately 50% of all ipsilateral GnRH neurones located throughout the DBB and hypothalamus. Few anterogradely labelled fibres were detected within the median eminence. Although such studies cannot define the presence of direct synaptic connections between VMN neurones and the GnRH cells, these observations support further the hypothesis that oestrogen-sensitive VMN neurones represent a direct transsynaptic input to the GnRH cell bodies which are involved in the generation of the LH surge in the ewe.     

Morphologic evidence that neurokinin B modulates gonadotropin-releasing hormone secretion via neurokinin 3 receptors in the rat median eminence

Recent studies suggest that arcuate neurokinin B (NKB) neurons play a role in the regulation of gonadotropin secretion, but there is little information on the relationship between these neurons and the hypothalamic reproductive axis. In the present study, dual-label fluorescent immunohistochemistry was used to visualize the relationship between gonadotropin-releasing hormone (GnRH) neurons and either proNKB or NK3 receptor (NK3R) immunoreactivity. Immunocytochemistry was also combined with i.p. injections of the fluorescent retrograde tracer aminostilbamidine to determine whether arcuate neuroendocrine neurons expressed either proNKB or NK3R. A dense interweaving and close apposition of GnRH and proNKB-immunoreactive (ir) fibers was observed within the rat median eminence, where GnRH axons expressed NK3R immunoreactivity. These data provide morphological evidence that NKB neurons could influence GnRH secretion via interaction with NK3R in the rat median eminence. Colocalization of GnRH and NK3R was also identified in fiber tracts converging within the organum vasculosum of the lamina terminalis. In contrast, only a small number (16%) of GnRH-ir somata exhibited NK3R staining. ProNKB and NK3R-ir somata were identified within the arcuate nucleus, but none of these neurons were labeled by aminostilbamidine. Thus, we found no evidence that arcuate NKB neurons project to the primary capillary plexus of the portal system. Arcuate neuroendocrine neurons, however, were surrounded and closely apposed by proNKB-ir puncta and fibers. These data suggest that NKB neurons could indirectly influence anterior pituitary function by inputs to arcuate neuroendocrine neurons, but through a receptor other than NK3R. Our results provide an anatomic framework for putative interactions between NKB neurons and the hypothalamic reproductive axis.     

Early development of forebrain gonadotrophin-releasing hormone (GnRH) neurones and the role of GnRH as an autocrine migration factor

Normal migration of the gonadotrophin-releasing hormone (GnRH) neurones during early development, from the olfactory region to the hypothalamus, is crucial for reproductive development in all vertebrates. The establishment of the GnRH system includes tangential migration of GnRH perikarya as well as extension of GnRH fibres to various areas of the central nervous system (CNS). The exact spatio-temporal nature of this process, as well as the factors governing it, are not fully understood. We studied the development of the GnRH system and the effects of GnRH knockdown using a newly developed GnRH3:EGFP transgenic zebrafish line. We found that enhanced green fluorescent protein is specifically and robustly expressed in GnRH3 neurones and fibres. GnRH3 fibres in zebrafish began to extend as early as 26 h post-fertilisation and by 4–5 days post-fertilisation had developed into an extensive network reaching the optic tract, telencephalon, hypothalamus, midbrain tegmentum and hindbrain. GnRH3 fibres also innervated the retina and projected into the trunk via the spinal cord. GnRH3 perikarya were observed migrating along their own fibres from the olfactory region to the preoptic area (POA) via the terminal nerve ganglion and the ventral telencephalon. GnRH3 cells were also observed in the trigeminal ganglion. The establishment of the GnRH3 fibre network was disrupted by morpholino-modified antisense oligonucleotides directed against GnRH3 causing abnormal fibre development and pathfinding, as well as anomalous GnRH3 perikarya localisation. These findings support the hypothesis that GnRH3 neurones migrate from the olfactory region to the POA and caudal hypothalamus. Novel data regarding the early development of the GnRH3 fibre network in the CNS and beyond are described. Moreover we show, in vivo , that GnRH3 is an important factor regulating GnRH3 fibre pathfinding and neurone localisation in an autocrine fashion.     

Evidence for estrogenic regulation of gonadotropin-releasing hormone neurons by glutamatergic neurons in the ewe brain: An immunohistochemical study using an antibody against vesicular glutamate transporter-2

Gonadotropin-releasing hormone (GnRH) secretion is controlled by various factors, including the excitatory neurotransmitter glutamate. Estrogen (E) regulates GnRH secretion by means of E-responsive cells in the brain that relay the feedback effects to the preoptic area (POA). We used an antibody to vesicular glutamate transporter 2 (VGluT2) to label glutamatergic neurons in the areas of the ewe brain that control GnRH secretion. VGluT2-immunoreactive cells were observed in the arcuate nucleus (ARC)/ventromedial hypothalamic nucleus (VMH) complex, POA, bed nucleus of stria terminalis (BnST), and A1 and A2 cell groups in the brainstem. In three ewes, E receptor-alpha was detected in 52-61% of glutamatergic neurons in ARC/VMH, 37-52% of neurons in the POA, and 37-58% of neurons in the BnST. E injection (i.m. or i.v.) increased the percentage of glutamatergic cells that expressed Fos protein in the ARC (P < 0.01 and P < 0.001, respectively). In six ewes, injection of the retrograde tracer Fluoro-Gold into the POA labeled cells in the ARC and 6-29% of these were also VGluT2-immunoreactive. Double-labeling of varicosities in the POA showed colocalization of VGluT2 in 12.5 +/- 3% of dopamine beta-hydroxylase-immunoreactive terminals, indicating that a subset of glutamatergic inputs could arise from brainstem noradrenergic neurons cells. In the POA, 60% of GnRH neurons had close appositions that were VGluT2-immunoreactive. We conclude that E-responsive glutamatergic neurons arising from the brainstem, the BnST, and ARC/VMH provide input to the POA and may be involved in the regulation of GnRH secretion.     

The gonadotropin-releasing hormone neurosecretory system of the jerboa (Jaculus orientalis) and its seasonal variations

The distribution of cells expressing gonadotropin-releasing hormone (GnRH) immunoreactivity was examined in the brain of adult jerboa during two distinct periods of the reproductive cycle. During spring-summer, when the jerboa is sexually active, a high density of cell bodies and fibres immunoreactive (IR) for GnRH was observed at the level of separation of the frontal lobes, in the medial septal nucleus (MS) and in the diagonal band of Broca (DBB), in the preoptic area (POA), in the organum vasculosum laminae terminalis (OVLT), in the retrochiasmatic area and hypothalamus. In autumn, when the jerboa is sexually inactive, GnRH-immunoreactivity was less intense than during spring-summer. In the POA, we noted a 55% decrease in the number of GnRH containing cells with no change in cell numbers in the MS-DBB. Furthermore, a lower density of GnRH immunopositive axon fibres is observed in all the previously mentioned structures and the immunoreaction intensity was very weak particularly within the median eminence and OVLT. Independently of the season, the GnRH immunoreactivity within neurones and fibres was similar in jerboas living in captivity and in jerboas living in their natural biotope. The effects of photoperiod on the density of POA-GnRH and arcuate nucleus beta-endorphin-containing cells were studied in jerboas maintained in long day [(LD) 16-h light, 8-h dark] and short day [(SD) 8-h light, 16-h dark] for 8 weeks. In the POA, the GnRH-IR cell number was not significantly altered by the photoperiod. Similarly, in the mediobasal hypothalamus, the number of beta-endorphin-IR neurones was not affected by such a parameter. Consequently, the GnRH seasonal variations cannot be correlated to changes in the photoperiod alone.