Modulation of Gonadotrophin-Releasing Hormone Pulse Generator Activity by the Pheromone in Small Ruminants

In small ruminants, such as goats and sheep, a primer pheromone produced by males induces an out-of-seasonal ovulation in anoestrous females, a phenomenon known as the male effect. The male effect is unique in that an external chemical stimulus can immediately modulate the activity of the hypothalamic gonadotrophin-releasing hormone (GnRH) pulse generator. We have established a monitoring method of the GnRH pulse generator activity in Shiba goat. Using this method as a sensitive bioassay to assess the male effect pheromone activity, we have shown that the male effect pheromone is synthesised in an androgen-dependent manner in the sebaceous glands or their vicinity in specific body regions in goats. Although chemical identity of the pheromone is yet to be determined, analyses of male goat hair extracts by gas chromatography fractionation suggest that the male effect pheromone is a volatile substance with relatively small molecular weight. From morphological and molecular biological studies in goats, it is suggested that the pheromone molecule is detected by a member of the V1R family located on both the olfactory neurones and the vomeronasal sensory neurones, and the pheromone signal is conveyed to the medial nucleus of amygdala via the main olfactory and vomeronasal pathways and, subsequently, to the hypothalamic GnRH pulse generator to enhance its activity.     

Effects of Glucose on Thyrotrophin-Releasing Hormone, Growth Hormone-Releasing Hormone, Somatostatin and Luteinizing Hormone-Releasing Hormone Release from Rat Hypothalamus in vitro

Increasing concentrations of D-glucose (1 to 25 mM) inhibited somatostatin, thyrotrophin-releasing hormone (TRH) and growth hormone-releasing hormone (GHRH) release from incubated adult rat hypothalami in a stereospecific manner. In contrast, the effects of D- and L-glucose on luteinizing hormone-releasing hormone release were virtually identical. Increasing concentrations of D-glucose also inhibited somatostatin release following depolarization with high K⁺, but had no obvious effect on depolarization-induced TRH or GHRH release when compared with L-glucose.
In conclusion, D-glucose exerts a potent, dose-related modulatory action on the release of rat hypothalamic TRH and GHRH as well as somatostatin in vitro. Further studies are required to establish any physiological relevance of glucose in the modulation of these hypothalamic neuropeptides.     

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.     

Glutamergic Action on Alpha-Melanocyte-Stimulating Hormone Release from the Rat Hypothalamus

Release of α-melanocyte-stimulating hormone (α-MSH) synthesized in the hypothalamus is regulated by monoaminergic neuronal systems. An endogenous dopaminergic system inhibits α-MSH release (1, 2) whilst serotoninergic systems exert a biphasic effect on peptide release (3). The toxic effects of neonatal peripheral administration of monosodium glutamate on hypothalamic neurons containing proopiomelanocortin- (POMC-) derived peptides (4, 5) suggest additionally the presence of glutamate receptors on or indirectly influencing the POMC neuron. By comparison of the effect of the excitatory amino-acid agonists N-methyl-D-aspartate (NMDA), quisqualate and kainate on the release of α-MSH from superfused slices of rat hypothalamus, we have demonstrated a stimulatory glutamergic action on α-MSH release mediated through NMDA-type receptors.     

Hypothalamic Effects of Tamoxifen on Oestrogen Regulation of Luteinising Hormone and Prolactin Secretion in Female Rats

Oestradiol (E₂) acts in the hypothalamus to regulate luteinising hormone (LH) and prolactin (PRL) secretion. Tamoxifen (TX) has been extensively used as a selective oestrogen receptor modulator, although its neuroendocrine effects remain poorly understood. In the present study, we investigated the hypothalamic effects of TX in rats under low or high circulating E₂ levels. Ovariectomised (OVX) rats treated with oil, E₂ or TX, or E₂ plus TX, were evaluated for hormonal secretion and immunohistochemical analyses in hypothalamic areas. Both E₂ and TX reduced LH levels, whereas TX blocked the E₂‐induced surges of LH and PRL. TX prevented the E₂‐induced expression of progesterone receptor (PR) in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC), although it did not alter PR expression in OVX rats. TX blocked the E₂ induction of c‐Fos in AVPV neurones, consistent with the suppression of LH surge. However, TX failed to prevent E₂ inhibition of kisspeptin expression in the ARC. In association with the blockade of PRL surge, TX increased the phosphorylation of tyrosine hydroxylase (TH) in the median eminence of OVX, E₂‐treated rats. TX also precluded the E₂‐induced increase in TH expression in the ARC. In all immunohistochemical analyses, TX treatment in OVX rats caused no measurable effect on the hypothalamus. Thus, TX is able to prevent the positive‐ but not negative‐feedback effect of E₂ on the hypothalamus. TX also blocks the effects of E₂ on tuberoinfundibular dopaminergic neurones and PRL secretion. These findings further characterise the anti‐oestrogenic actions of TX in the hypothalamus and provide new information on the oestrogenic regulation of LH and PRL.     

Male Effect Pheromone Tickles the Gonadotrophin‐Releasing Hormone Pulse Generator

In sheep and goats, the primer pheromone produced by the male induces out‐of‐seasonal ovulation in anoestrous females, the so‐called ‘male effect.’ Because the initial endocrine event following reception of the pheromone is the stimulation of pulsatile luteinising hormone (LH) secretion, the central target of the pheromone is considered to be the putative gonadotrophin‐releasing hormone (GnRH) pulse generator. Using electrophysiological techniques to record multiple‐unit activity (MUA) in close proximity to kisspeptin neurones in the arcuate nucleus (ARC) of Shiba goats, we found that bursts (volleys) of MUA occur at regular intervals, and repetitive bursts are invariably associated with discrete pulses of LH, suggesting that the ARC kisspeptin neurones may be the intrinsic source of the GnRH pulse generator. A brief exposure of female goats to the pheromone immediately elicited an instantaneous rise in MUA, which is followed by an MUA volley and an accompanying LH pulse, indicating that the pheromone signal is transmitted to a subset of the ARC kisspeptin neurones to activate them. Because it has been suggested that the neurokinin B and dynorphin coexpressed in those neurones play critical roles in generating rhythmic bursts, they may be involved in the intracellular pheromone actions that are responsible for inducing the GnRH pulse.     

Chronic Exposure to Low Levels of Oestradiol-17β Affects Oestrous Cyclicity, Hypothalamic Norepinephrine and Serum Luteinising Hormone in Young Intact Rats

Chronic exposure to oestrogens is known to inhibit the secretion of luteinising hormone (LH) in rats, leading to anovulation. Hypothalamic catecholamines, norepinephrine and dopamine play an important role in LH regulation. However, the effects of chronic exposure to low levels of oestradiol on hypothalamic catecholamines have not been investigated thoroughly. In the present study, adult female Sprague–Dawley rats were either sham implanted or implanted with 17β‐oestradiol (E₂) pellets (20 ng/day) for 30 (E‐30), 60 (E‐60) or 90 (E‐90) days. E₂ exposure affected oestrous cyclicity and ovarian morphology in a duration‐dependent manner. There was no change in oestrous cyclicity in E‐30 rats; however, 75% of E‐60 and 95% of E‐90 rats were acyclic (P < 0.05). Cycling rats from E‐30 or the control group were killed at different time points on the afternoon of pro‐oestrous. E‐30 rats in oestrous, constant oestrous rats in the E‐60 and E‐90 groups and a group of old constant oestrous (OCE) rats were killed at 12.00 h. LH was measured in the serum by radioimmunoassay. Individual hypothalamic nuclei that are involved in LH regulation were microdissected and analysed for norepinephrine and dopamine levels using high‐performance liquid chromatography/electrochemical detection. Norepinephrine levels in the hypothalamic nuclei increased significantly in control and E‐30 groups during the afternoon of pro‐oestrous, which was accompanied by a rise in LH levels (P < 0.05). On the day of oestrous, norepinephrine concentrations in hypothalamic nuclei and serum LH were significantly lower in E‐60, E‐90 and OCE rats compared to E‐30 and control rats. On the other hand, dopamine levels declined significantly in one hypothalamic nucleus. These results indicate that chronic E₂ exposure affects hypothalamic catecholamine and serum LH levels in a duration‐dependent manner. This coincides well with the loss of cyclicity observed in these animals. These results suggest that repeated exposure to endogenous oestrogens could play a role in reproductive senescence.     

Kisspeptin/Metastin: A Key Molecule Controlling Two Modes of Gonadotrophin-Releasing Hormone/Luteinising Hormone Release in Female Rats

Kisspeptin (also known as metastin), a hypothalamic peptide, has attracted attention as a key molecule in the release of gonadotrophin-releasing hormone (GnRH) in various mammalian species, such as rodents, sheep and primates. Two populations of kisspeptin neurones in the brain may control two modes of GnRH release to time the onset of puberty and regulate oestrous cyclicity in rats and mice. One population of kisspeptin neurones, located in the anteroventral periventricular nucleus, appears to be responsible for the induction of the GnRH surge that leads to the luteinising hormone surge and ovulation. The other, located in the hypothalamic arcuate nucleus, appears to be involved in generating GnRH pulses, resulting in luteinising hormone pulses followed by follicular development and steroidogenesis in the ovary. The present review focuses on the physiological role of the two populations of kisspeptin neurones in controlling gonadal functions by generating the two modes of GnRH release in a female rat model.     

Evidence for Oestrogen Receptor α-Immunoreactivity in Gonadotrophin-Releasing Hormone-Expressing Neurones

Having used the cingulate cortex to demonstrate the validity of our methods for detecting hitherto unrecognized oestrogen receptor alpha (ERalpha)-immunoreactive neurones, we have now employed immunoprecipitation and double-label immunohistochemistry to investigate whether the ERalpha protein is present in gonadotrophin-releasing hormone (GnRH)-containing cells. The immortalized GnRH cell line GT1-7 and GnRH neurones within the rat preoptic area were found to possess ERalpha-immunoreactivity (ERalpha-IR). These observations indicate that oestrogen may regulate the synthesis and release of GnRH by direct actions on GnRH neurones.     

Kisspeptin Regulates Tuberoinfundibular Dopaminergic Neurones and Prolactin Secretion in an Oestradiol‐Dependent Manner in Male and Female Rats

Prolactin (PRL) secretion is inhibited by hypothalamic dopamine. Kisspeptin controls luteinising hormone (LH) secretion and is also involved in PRL regulation. We further investigated the effect of kisspeptin‐10 (Kp‐10) on the activity of tuberoinfundibular dopaminergic (TIDA) neurones and the role of oestradiol (E₂) in this mechanism. Female and male rats were injected with i.c.v. Kp‐10 and evaluated for PRL release and the activity of dopamine terminals in the median eminence (ME) and neurointermediate lobe of the pituitary (NIL). Kp‐10 at the doses of 0.6 and 3 nmol increased plasma PRL and decreased 4‐dihydroxyphenylacetic acid (DOPAC) levels in the ME and NIL of ovariectomised (OVX), E₂‐treated rats but had no effect in OVX. In gonad‐intact males, 3 nmol Kp‐10 increased PRL secretion and decreased DOPAC levels in the ME but not in the NIL. Castrated males treated with either testosterone or E₂ also displayed increased PRL secretion and reduced ME DOPAC in response to Kp‐10, whereas castrated rats receiving oil or dihydrotestosterone were unresponsive. By contrast, the LH response to Kp‐10 was not E₂‐dependent in either females or males. Additionally, immunohistochemical double‐labelling demonstrated that TIDA neurones of male rats contain oestrogen receptor (ER)‐α, with a higher proportion of neurones expressing ERα than in dioestrous females. The dopaminergic neurones of periventricular hypothalamic nucleus displayed much lower ERα expression. Thus, TIDA neurones express ERα in male and female rats, and kisspeptin increases PRL secretion through inhibition of TIDA neurones in an E₂‐dependent manner in both sexes. These findings provide new evidence about the role of kisspeptin in the regulation of dopamine and PRL.     

Oestrogen-dependent suppression of pulsatile luteinising hormone secretion and kiss1 mRNA expression in the arcuate nucleus during late lactation in rats

Follicular development and ovulation are strongly suppressed during lactation in mammals via a profound suppression of gonadotrophin secretion. The present study aimed to examine the role of oestrogen feedback action in suppressing luteinising hormone (LH) secretion and hypothalamic kisspeptin expression during the latter half of lactation. Plasma LH concentrations kept at low levels throughout the lactating period in intact and oestrogen‐replaced ovariectomised (OVX) lactating rats, whereas plasma LH concentrations gradually elevated from day 10 postpartum in lactating OVX rats. OVX lactating rats showed frequent LH pulses at late lactation, although the LH pulses were significantly inhibited by an oestrogen replacement, which is much less effective on LH release in nonlactating rats. Oestrogen replacement in lactating OVX rats significantly reduced the number of Kiss1 mRNA‐expressing cells in the arcuate nucleus (ARC) at late lactation, although the same oestrogen treatment did not affect the number of Kiss1‐expressing cells in nonlactating controls. Exogenous kisspeptin challenge (0.2 nmol) into the third cerebroventricle significantly increased LH secretion in lactating OVX, lactating OVX + subcutaneous 17β‐oestradiol and intact lactating rats at day 16 postpartum. These results suggest that LH pulse suppression during late lactation could be a result of the enhanced oestrogen‐dependent suppression of ARC kisspeptin expression.     

Coexistence of Gonadotrophin-Releasing Hormone and Galanin: Immunohisto-chemical and Functional Studies

Our observation of galanin-immunoreactive perikarya with morphological characteristics strikingly similar to those containing gorvadotrophin-releasmg hormone (GnRH) led us to undertake double-label immunohistochemical studies on the distribution of these peptides. A considerable proportion of GnRH-containing perikarya in the rat preoptic area were found to be immunoreactive for galanin. In order to establish whether this coexistence contributes to the control of luteinizing hormone (LH) release, the two peptides have been perfused alone and in combination through anterior pituitary cell columns derived from female rats in various endocrine conditions. Galanin at 0.1 μ M had a small stimulatory effect on LH release from cells obtained on the day of pro-oestrus; this effect was less than that obtained with GnRH at 4 nM. Cells from oestrous rats showed no LH release in response to galanin. The studies undertaken thusfar have not indicated that galanin has a priming effect on the release of LH induced by subsequent treatment with galanin or a potentiating effect when administered together with GnRH. Delta sleep-inducing peptide, another neuropeptide recently reported to be coexistent with GnRH, was also found to be capable of inducing a small discharge of LH from anterior pituitary cells obtained on the day of pro-oestrus. Further studies will be required to establish the full consequences of the coexistence of these peptides and GnRH.     

Differential Expression of RFamide‐Related Peptide,a Mammalian Gonadotrophin‐Inhibitory Hormone Orthologue,and Kisspeptin in the Hypothalamus of Abadeh Ecotype Does During Breeding and Anoestrous Seasons

Gonadotrophin‐inhibitory hormone (GnIH) is a novel hypothalamic neuropeptide that was discovered in birds as an inhibitory factor for gonadotrophin release. RFamide‐related peptide (RFRP) is a mammalian GnIH orthologue that inhibits gonadotrophin synthesis and release in mammals through actions on gonadotrophin‐releasing hormone (GnRH) neurones and gonadotrophs, mediated via the GnIH receptor (GnIH‐R), GPR147. On the other hand, hypothalamic kisspeptin provokes the release of GnRH from the hypothalamus. The present study aimed to compare the expression of RFRP in the dorsomedial hypothalamus and paraventricular nucleus (DMH/PVN) and that of kisspeptin in the arcuate nucleus (ARC) of the female goat hypothalamus during anoestrous and breeding seasons. Mature female Abadeh does were used during anoestrus, as well as the follicular and luteal phases of the cycle. The number of RFRP‐immunoreactive (‐IR) neurones in the follicular phase was lower than in the luteal and anoestrous stages. Irrespective of the ovarian stage, the number of RFRP‐IR neurones in the rostral and middle regions of the DMH/PVN was higher than in the caudal region. By contrast, the number of kisspeptin‐IR neurones in the follicular stage was greater than in the luteal stage and during the anoestrous stage. Irrespective of the stage of the ovarian cycle, the number of kisspeptin‐IR neurones in the caudal region of the ARC was greater than in the middle and rostral regions. In conclusion, RFRP‐IR cells were more abundant in the rostral region of the DMH/PVN nuclei of the hypothalamus, with a greater number being found during the luteal and anoestrous stages compared to the follicular stage. On the other hand, kisspeptin‐IR neurones were more abundant in the caudal part of the ARC, with a greater number recorded in the follicular stage compared to the luteal and anoestrous stages.     

Adrenomedullectomy Prevents the Suppression of Pulsatile Luteinising Hormone Release During Fasting in Female Rats

Fasting inhibits the pulsatile secretion of luteinising hormone (LH) in female rats, an effect which is potentiated by the presence of oestradiol (E2). We have previously described various pharmacological or surgical treatments that can rapidly restore the pulses in a fasting animal. Nevertheless, the central and peripheral mechanisms that mediate this suppression of the pulses remain unclear. We have recently shown that adrenomedullectomy prevents the suppression of LH pulses by insulin-induced hypoglycaemia, a state which activates the sympathoadrenal axis. The present study was undertaken to establish whether this axis might contribute to the loss of the pulses that occurs in ovariectomised E2-treated rats that have been fasted for 48 h. Following sham adrenomedullectomy LH pulses were observed in animals fed ad libitum; after 48 h of fasting the animals that had received this sham procedure showed a significant suppression of LH levels and LH pulse frequency. In contrast, adrenomedullectomy prevented the inhibition of the pulses by 48 h of fasting; it had no effect on the pulses in the absence of fasting. These results suggest that adrenomedullary activity plays a significant role in the fasting-induced suppression of LH pulses in rats.     

Hypothalamic Galanin‐Like Peptide Rescues the Onset of Puberty in Food‐Restricted Weanling Rats

Galanin‐like peptide (GALP) is a known mediator of metabolism and reproduction; however, the role that GALP plays in the onset of puberty is unknown. First, we tested the hypothesis that central GALP administration could rescue puberty in food‐restricted weanling rats. GALP treatment in food‐restricted rats of both sexes rescued the timing of the onset of puberty to that seen in ad lib. fed controls. Second, we tested whether GALP translation knocked‐down in ad lib. fed, prepubertal rats would alter the timing of puberty. Knock‐down females, but not males, showed a significant (P < 0.01) delay in the onset of puberty compared to controls. Third, we sought evidence that the role of GALP in pubertal onset is mediated by the kisspeptin system. In situ hybridisation analyses showed a significant (P < 0.01) reduction in Kiss1 mRNA within the hypothalamic arcuate nucleus in food‐restricted rats compared to ad lib. fed controls and this reduction was prevented with i.c.v. GALP administration. Furthermore, analyses of Fos‐immunoreactivity (‐IR) after i.c.v. GALP treatment did not elicit Fos‐IR within any kisspeptin neurones, nor are GALP and kisspeptin peptides or mRNA colocalised. These data demonstrate that hypothalamic GALP infusion maintained the onset of puberty in food‐restricted weanling rats, although probably not via direct innervation of kisspeptin neurones.     

Gonadotrophin‐Inhibitory Hormone in the Cichlid Fish Cichlasoma dimerus: Structure,Brain Distribution and Differential Effects on the Secretion of Gonadotrophins and Growth Hormone

The role of gonadotrophin‐inhibitory hormone (GnIH) in the inhibition of the reproductive axis has been well‐established in birds and mammals. However, its role in other vertebrates, such as the teleost fish, remains controversial. In this context, the present study aimed to evaluate whether GnIH modulates the release of gonadotrophins and growth hormone (GH) in the cichlid fish Cichlasoma dimerus. First, we partially sequenced the precursor polypeptide for GnIH and identified three putative GnIH peptides. Next, we analysed the expression of this precursor polypeptide via a polymerase chain reaction in the reproductive axis of both sexes. We found a high expression of the polypeptide in the hypothalamus and gonads of males. Immunocytochemistry allowed the observation of GnIH‐immunoreactive somata in the nucleus posterioris periventricularis and the nucleus olfacto‐retinalis, with no differences between the sexes. GnIH‐immunoreactive fibres were present in all brain regions, with a high density in the nucleus lateralis tuberis and at both sides of the third ventricle. Finally, we performed in vitro studies on intact pituitary cultures to evaluate the effect of two doses (10^?6 m and 10^?8 m ) of synthetic C. dimerus (cd‐) LPQRFa‐1 and LPQRFa‐2 on the release of gonadotrophins and GH. We observed that cd‐LPQRFa‐1 decreased β‐luteinising hormone (LH) and β‐follicle‐stimulating hormone (FSH) and also increased GH release to the culture medium. The release of β‐FSH was increased only when it was stimulated with the higher cd‐LPQRFa‐2 dose. The results of the present study indicate that cd‐LPQRFa‐1, the cichlid fish GnIH, inhibits β‐LH and β‐FSH release and stimulates GH release in intact pituitary cultures of C. dimerus. The results also show that cd‐LPQRF‐2 could act as an β‐FSH‐releasing factor in this fish species.     

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.