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.     

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.     

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.     

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.     

Neurokinin 3 Receptor Immunoreactivity in the Septal Region, Preoptic Area and Hypothalamus of the Female Sheep: Colocalisation in Neurokinin B Cells of the Arcuate Nucleus but not in Gonadotrophin-Releasing Hormone Neurones

Recent evidence has implicated neurokinin B (NKB) in the complex neuronal network mediating the effects of gonadal steroids on the regulation of gonadotrophin-releasing hormone (GnRH) secretion. Because the neurokinin 3 receptor (NK3R) is considered to mediate the effects of NKB at the cellular level, we determined the distribution of immunoreactive NK3R in the septal region, preoptic area (POA) and hypothalamus of the ewe. NK3R cells and/or fibres were found in areas including the bed nucleus of the stria terminalis, POA, anterior hypothalamic and perifornical areas, dopaminergic A15 region, dorsomedial and lateral hypothalamus, arcuate nucleus (ARC) and the ventral premammillary nucleus. We also used dual-label immunocytochemistry to determine whether a neuroanatomical basis for direct modulation of GnRH neurones by NKB was evident. No GnRH neurones at any rostral-caudal level were observed to contain NK3R immunoreactivity, although GnRH neurones and fibres were in proximity to NK3R-containing fibres. Because NKB fibres formed close contacts with NKB neurones in the ARC, we determined whether these NKB neurones also contained immunoreactive NK3R. In luteal-phase ewes, 64% ± 11 of NKB neurones colocalised NK3R. In summary, NK3R is distributed in areas of the sheep POA and hypothalamus known to be involved in the control of reproductive neuroendocrine function. Colocalisation of NK3R in NKB neurones of the ARC suggests a potential mechanism for the autoregulation of this subpopulation; however, the lack of NK3R in GnRH neurones suggests that the actions of NKB on GnRH neurosecretory activity in the ewe are mediated indirectly via other neurones and/or neuropeptides.     

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.     

Oestrogen‐Induced Activation of Preoptic Kisspeptin Neurones May be Involved in the Luteinising Hormone Surge in Male and Female Japanese Monkeys

The oestrogen‐induced luteinising hormone (LH) surge is evident in male primates, including humans, whereas male rodents never show the LH surge, even when treated with a preovulatory level of oestrogen. This suggests that the central mechanism governing reproductive hormones in primates is different from that in rodents. The present study aimed to investigate whether male Japanese monkeys conserve a brain mechanism mediating the oestrogen‐induced LH surge via activation of kisspeptin neurones. Adult male and female Japanese monkeys were gonadectomised and then were treated with oestradiol‐17β for 2 weeks followed by a bolus injection of oestradiol benzoate. Both male and female monkeys showed an oestrogen‐induced LH surge. In gonadectomised monkeys sacrificed just before the anticipated time of the LH surge, oestrogen treatment significantly increased the number of KISS1‐expressing cells in the preoptic area (POA) and enhanced the expression of c‐fos in POA KISS1‐positive cells of males and females. The oestrogen treatment failed to induce c‐fos expression in the arcuate nucleus (ARC) kisspeptin neurones in both sexes just prior to LH surge onset. Thus, kisspeptin neurones in the POA but not in the ARC might be involved in the positive‐feedback action of oestrogen that induces LH surge in male Japanese monkeys, as well as female monkeys. The present results indicate that oestrogen‐induced activation of POA kisspeptin neurones may contribute to the LH surge generation in both sexes. The conservation of the LH surge generating system found in adult male primates, unlike rodents, could be a result of the capability of oestrogen to induce POA kisspeptin expression and activation.     

Neural Systems Mediating Seasonal Breeding in the Ewe

Seasonal reproduction in ewes is caused by a dramatic increase in response to oestradiol (E₂) negative feedback during the nonbreeding (anoestrous) season. Considerable evidence supports the hypothesis that A15 dopaminergic neurones in the retrochiasmatic area (RCh) play a key role in these seasonal changes. These A15 neurones are stimulated by E₂ and inhibit gonadotrophin‐releasing hormone (GnRH) secretion in anoestrus, but not the breeding season. Because A15 neurones do not contain oestrogen receptors‐α (ERα), it is likely that E₂‐responsive afferents stimulate their activity when circulating E₂ levels increase during anoestrus. Retrograde tract tracing studies identified a limited set of ERα‐containing afferents primarily found in four areas [ventromedial preoptic area, RCh, ventromedial and arcuate (ARC) nuclei]. Pharmacological and anatomical data are consistent with GABA‐ and glutamate‐containing afferents controlling A15 activity in anoestrus, with E₂ inhibiting GABA and stimulating glutamate release at this time of year. Tract tracing demonstrated that A15 efferents project posteriorly to the median eminence and the ARC, suggesting possible direct actions on GnRH terminals or indirect actions via kisspeptin neurones in the ARC to inhibit GnRH in anoestrus. Identification of this neural circuitry sets the stage for the development of specific hypotheses for morphological or transmitter/receptor expression changes that would account for seasonal breeding in ewes.     

Kisspeptin and the Preovulatory Gonadotrophin‐Releasing Hormone/Luteinising Hormone Surge in the Ewe: Basic Aspects and Potential Applications in the Control of Ovulation

The identification of the neural mechanisms controlling ovulation in mammals has long been a ‘holy grail’ over recent decades, although the recent discovery of the kisspeptin systems has totally changed our views on this subject. Kisspeptin cells are the major link between gonadal steroids and gonadotrophin‐releasing hormone (GnRH) neurones. In the female rodent, kisspeptin cells of the preoptic area are involved in the positive‐feedback action of oestrogen on GnRH secretion, although the picture appears more complicated in the ewe. As in rodents, activation of preoptic kisspeptin neurones accompanies the GnRH surge in the ewe but an active role for arcuate kisspeptin neurones has also been proposed. Experimentally, kisspeptin is able to restore reproductive function when the hypothalamic‐hypophyseal ovarian axis is quiescent. For example, i.v. infusion of a low dose of peptide in anoestrous ewes induces an immediate and sustained release of gonadotrophin, which subsides and then provokes a luteinising hormone (LH) surge a few hours later. This pharmacological intervention induces the same hormonal changes normally observed during the follicular phase of the oestrous cycle, including the secretion of oestrogen and its negative‐ and positive‐feedback actions on the secretion of LH and follicle‐stimulating hormone. Accordingly, a high percentage of kisspeptin‐infused animals ovulated. Although the multiple facets of how the kisspeptin systems modulate GnRH secretion are not totally understood, the demonstration that exogenous kisspeptin administration can induce ovulation in anovulatory animals paves the way for future therapeutic applications aiming to control reproduction.     

The Luteinising Hormone Surge‐Generating System is Functional in Male Goats as in Females: Involvement of Kisspeptin Neurones in the Medial Preoptic Area

A luteinising hormone (LH) surge is fundamental to the induction of ovulation in mammalian females. The administration of a preovulatory level of oestrogen evokes an LH surge in ovariectomised females, whereas the response to oestrogen in castrated males differs among species; namely, the LH surge‐generating system is sexually differentiated in some species (e.g. rodents and sheep) but not in others (e.g. primates). In the present study, we aimed to determine whether there is a functional LH surge‐generating system in male goats, and whether hypothalamic kisspeptin neurones in male goats are involved in the regulation of surge‐like LH secretion. By i.v. infusion of oestradiol (E₂; 6 μg/h) for 16 h, a surge‐like LH increase occurred in both castrated male and ovariectomised female goats, although the mean peak LH concentration was lower and the mean peak of the LH surge was later in males compared to females. Dual staining with KISS1 in situ hybridisation and c‐Fos immunohistochemistry revealed that E₂ treatment significantly increased c‐Fos expression in the medial preoptic area (mPOA) KISS1 cells in castrated males, as well as ovariectomised females. By contrast, dual‐labelled cells were scarcely detected in the arcuate nucleus (ARC) after E₂ treatment in both sexes. These data suggest that kisspeptin neurones in the mPOA, but not those in the ARC, are involved in the induction of surge‐like LH secretion in both male and female goats. In summary, our data show that the mechanism that initiates the LH surge in response to oestrogen, the mPOA kisspeptin neurones, is functional in male goats. Thus, sexual differentiation of the LH surge‐generating system would not be applicable to goats.     

Central injection of senktide, an NK3 receptor agonist, or neuropeptide Y inhibits LH secretion and induces different patterns of Fos expression in the rat hypothalamus

Arcuate neurokinin B (NKB) neurons express estrogen receptor-alpha and are strongly modulated by gonadal steroids. Although numerous studies suggest that NKB neurons participate in the reproductive axis, there is no information on the regulation of luteinizing hormone (LH) secretion by NKB or its receptor, NK3. In the present study, we determined if central injection of senktide, a selective NK3 receptor agonist, would alter serum LH in ovariectomized, estrogen-primed rats. The effects of senktide were compared to neuropeptide Y (NPY), a well-characterized modulator of LH secretion. Saline, senktide, or NPY was injected into the lateral ventricle of unanesthetized rats and serial blood samples were collected for LH radioimmunoassay. The rats were sacrificed 90 min after injection and the brains were removed and processed for Fos immunocytochemistry. A significant inhibition of serum LH was observed from 30 to 90 min after injection of senktide relative to saline controls. In the senktide-injected rats, the inhibition of serum LH was accompanied by increased Fos expression in the medial preoptic area and arcuate nucleus--two reproductive control centers. Senktide also induced Fos in the paraventricular nuclei (PVN) and supraoptic nuclei (SON). Injection of NPY also inhibited serum LH but increased Fos expression only in the PVN and SON. This study provides the first demonstration of alterations in LH secretion by an NK3 receptor agonist. These data, combined with the induction of Fos in medial preoptic and arcuate neurons, strongly support the hypothesis that NKB neurons play a role in the regulation of gonadotropin secretion.     

Progesterone can block the preovulatory gonadotropin-releasing hormone/luteinising hormone surge in the ewe by a direct inhibitory action on oestradiol-responsive cells within the hypothalamus

Elevated oestradiol concentrations during the follicular phase stimulate a surge in gonadotropin-releasing hormone (GnRH) and luteinising hormone (LH) concentrations, which leads to ovulation. Progesterone can block the oestradiol-induced GnRH/LH surge, but the mechanism that is involved is unclear. We examined the effect of progesterone on oestradiol-induced activation of cells within the ovine hypothalamus/preoptic area (POA) to determine: (i) in which regions progesterone acts to block the GnRH/LH surge and (ii) whether progesterone directly or indirectly prevents activation of oestradiol-responsive cells. Cellular activation was assessed by measuring the number of cells that expressed Fos (an immediate early gene). Exposure to increased oestradiol concentrations in the absence of progesterone (which normally stimulates a LH surge) did not cause any region-specific changes in hypothalamic Fos expression during the activation stage of the LH surge-induction process (Experiment 1). The same treatment significantly increased cellular activation within the POA, lateral septum (LS), and arcuate nucleus at the time of surge onset (Experiment 2). Concurrent exposure to increased oestradiol and progesterone concentrations during the activation stage of the surge-induction process (which normally blocks the LH surge) was associated with significantly reduced cellular activation within the ventromedial hypothalamus and anterior hypothalamic area, relative to the positive controls (oestradiol increment alone) and arcuate nucleus relative to the negative controls (no increment in oestradiol) during the activation stage (Experiment 1). At the time of surge onset (Experiment 2), exposure to progesterone during the activation period prevented the oestradiol-induced increase in cellular activation that occurred in the POA, LS and arcuate nucleus of the positive controls. These results demonstrated that oestradiol and progesterone induced differential region- and time-specific effects on cellular activation within the regions of the ovine brain that generate the preovulatory GnRH/LH surge. Moreover, the lack of cellular activation within the POA, LS and arcuate nucleus at the time of surge onset in animals exposed to progesterone during the activation stage is consistent with the hypothesis that progesterone can block the preovulatory surge by direct inhibition of oestradiol-induced cellular activation in these areas.     

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.     

Aromatase knockout mice show normal steroid-induced activation of gonadotrophin-releasing hormone neurones and luteinising hormone surges with a reduced population of kisspeptin neurones in the rostral hypothalamus

We recently reported that female aromatase knockout (ArKO) mice show deficits in sexual behaviour and a decreased population of kisspeptin‐immunoreactive neurones in the rostral periventricular area of the third ventricle (RP3V), resurrecting the question of whether oestradiol actively contributes to female‐typical sexual differentiation. To further address this question, we assessed the capacity of ArKO mice to generate a steroid‐induced luteinising hormone (LH) surge. Adult, gonadectomised wild‐type (WT) and ArKO mice were given silastic oestradiol implants s.c. and, 1 week later, received s.c. injections of either oestradiol benzoate (EB) followed by progesterone, EB alone, or no additional steroids to activate gonadotrophin‐releasing hormone (GnRH) neurones and generate an LH surge. Treatment with EB and progesterone induced significant Fos/GnRH double‐labelling and, consequently, an LH surge in female WT and in ArKO mice of both sexes but not in male WT mice. ArKO mice of both sexes had fewer cells expressing Kiss‐1 mRNA in the RP3V compared to female WT mice but had more Kiss‐1 mRNA‐expressing cells compared to WT males, reflecting an incomplete sexual differentiation of this system. To determine the number of cells expressing kisspeptin, the same experimental design was repeated in Experiment 2 with the addition of groups of WT and ArKO mice that were given EB + progesterone and sacrificed 2 h before the expected LH surge. No differences were observed in the number of kisspeptin‐immunoreactive cells 2 h before and at the time of the LH surge. The finding that ArKO mice of both sexes have a competent LH surge system suggests that oestradiol has predominantly defeminising actions on the GnRH/LH surge system in males and that the steroid‐induced LH surge can occur in females even with a greatly reduced population of kisspeptin neurones in the RP3V.     

Gonadotrophin-Releasing Hormone Pulse Generator Activity in the Hypothalamus of the Goat

Pulsatile release of gonadotrophin-releasing hormone (GnRH) is indispensable to maintain normal gonadotrophin secretion. The pulsatile secretion of GnRH is associated with synchronised electrical activity in the mediobasal hypothalamus (i.e. multiple unit activity; MUA), which is considered to reflect the rhythmic oscillations in the activity of the neuronal network that drives pulsatile GnRH secretion. However, the cellular source of this ultradian rhythm in GnRH activity is unknown. Direct input from kisspeptin neurones in the arcuate nucleus (ARC) to GnRH cell bodies in the medial preoptic area or their terminals in the median eminence could be the intrinsic source for driving the GnRH pulse generator. To determine whether kisspeptin signalling could be responsible for producing pulsatile GnRH secretion, we studied goats, measured plasma levels of luteinising hormone (LH) and recorded MUA in the posterior ARC, where the majority of kisspeptin neuronal cell bodies are located. Rhythmic volleys of MUA were found to be accompanied by LH pulses with regular intervals in the ARC, where kisspeptin neuronal cell bodies were found. Exogenous administration of kisspeptin stimulated a sustained increase in LH secretion, without influencing MUA, suggesting that the GnRH pulse generator, as reflected by MUA, originated from outside of the network of GnRH neurones, and could plausibly reflect the pacemaker activity of kisspeptin neurones, whose projections reach the median eminence where GnRH fibres project. These observations suggest that the kisspeptin neurones in the ARC may be the intrinsic source of the GnRH pulse generator.     

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

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

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.     

Inhibition of sexual behaviour and the luteinizing hormone surge by intracerebral progesterone implants in the female sheep

In female sheep, progesterone blocks the induction by oestradiol of both sexual behaviour and the pre-ovulatory surges of gonadotrophin releasing hormone (GnRH) and luteinising hormone (LH). However, the central sites of action of progesterone remain poorly defined, so we attempted to locate them by implanting progesterone intracerebrally in ovariectomised ewes treated with exogenous steroids to induce oestrous behaviour and the LH surge. Single bilateral implants or a double bilateral implants filled with progesterone or cholesterol were placed in the ventromedial hypothalamus (VMH) or the preoptic area (POA). Control ewes were not implanted. To determine the inhibitory capacity of the central progesterone implants, ewes received an injection (i.m.) of 8 μg or 16 μg of oestradiol. The single bilateral implants of progesterone failed to block oestrous behaviour and the LH surge induced by 8 μg of oestradiol. Double bilateral progesterone implants in the VMH blocked the sexual behaviour (P < 0.05) and the LH surge (P < 0.05), but implants in the POA blocked only sexual receptivity (P < 0.05). No changes were observed after central implantation of cholesterol. Our results support the hypothesis that progesterone acts centrally in the VMH and the POA to inhibit the induction of LH surge and sexual behaviour by oestradiol.     

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.