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.     

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.     

Activity of Hypothalamic Dopaminergic Neurones During the Day of Oestrus: Involvement in Prolactin Secretion

A secretory surge of prolactin occurs on the afternoon of oestrus in cycling rats. Pituitary prolactin is inhibited by dopamine. We evaluated the activity of the neuroendocrine dopaminergic neurones during oestrus and dioestrus, as determined by dopaminergic activity in the median eminence and neurointermediate lobe of the pituitary, as well as Fos‐related antigen expression in tyrosine hydroxylase (TH)‐immunoreactive (ir) neurones of the arcuate nucleus (ARC) and periventricular nucleus (Pe). During oestrus, the 4‐dihydroxyphenylacetic acid/dopamine ratio in the median eminence decreased at 16.00 h, coinciding with the increase in plasma prolactin levels. Similarly, the expression of Fos‐related antigen in TH‐ir neurones of Pe and rostral‐, dorsomedial‐ and caudal‐ARC also decreased at 16.00 h. On dioestrus, 4‐dihydroxyphenylacetic acid/dopamine ratio in the median eminence and Fos‐related antigen expression in TH‐ir neurones of Pe and rostral‐ARC decreased at 18.00 h, whereas prolactin levels were unaltered. No variation in dopaminergic activity was found in the neurointermediate lobe of the pituitary on either oestrus or dioestrus. The number of TH‐ir neurones in the ARC and parameters of dopaminergic activity were found to be generally lower on oestrus compared to dioestrus. The transitory decrease in the activity of neuroendocrine dopaminergic neurones temporally associated with the prolactin surge on the afternoon of oestrus suggests a role for dopamine in the generation of the oestrous prolactin surge.     

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.     

Oestrogen Receptor-α-Immunoreactive Neurones Project to the Suprachiasmatic Nucleus of the Female Syrian Hamster

Ovarian steroid hormones regulate circadian period and phase, but classical receptors for these hormones are absent in the circadian pacemaker localized in the suprachiasmatic nucleus of the hypothalamus (SCN). In order to determine whether effects of oestrogen may be exerted through steroid-binding systems afferent to the SCN we have performed double label immunocytochemistry for oestrogen receptor-alpha(ER-alpha) and the retrograde tracer cholera toxin B subunit (CtB) after its application to the SCN. Most of the areas that contain ER-alpha-immunoreactive (ERalpha-ir) cells also contained cells afferent to the SCN. The percentage of neurones afferent to the SCN which show ERalpha-immunoreactivity varies between areas. As many as one-third of the neurones afferent to the SCN in some parts of the preoptic area and the corticomedial amygdala are ERalpha-ir. Very few of the afferent neurones from the septum and the central grey are ERalpha-ir, whereas an intermediate proportion of afferents from the bed nucleus of the stria terminalis and the arcuate nucleus are ERalpha-ir. Our retrograde tracing results were compared with results of anterograde tracing from some of the sites containing SCN afferents. Using a combined retrograde and anterograde tracing technique we tested the possibility that single ERalpha-ir neurones afferent to the SCN could receive reciprocal innervation by SCN efferents. Although we found SCN input to some SCN afferent neurones, we found no evidence of reciprocity between single ERalpha-ir cells and the SCN. Our results indicate the existence of oestrogen binding systems afferent to the SCN. These neuroanatomical pathways may mediate effects of gonadal steroid hormones on circadian rhythms.     

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.     

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.     

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.     

Possible Role of Neurokinin A in the Control of Prolactin Secretion in Rats and Hamsters

The possible role of neurokinin A (NKA) in the control of prolactin secretion was studied in vivo, by injecting anti-NKA serum to ovariectomized rats treated with estrogens and to proestrous rats and hamsters. Injections of an anti-NKA serum to ovariectomized rats treated with two doses of 80 μg 17ß-estradiol 24 h apart, or treated chronically with estradiol implants induced a significant decrease of serum prolactin levels as compared with those of similarly treated rats injected with normal rabbit serum. In proestrous rats, the anti-NKA serum did not modify the afternoon surge of prolactin or luteinizing hormone, but when the antiserum was injected the day before, on diestrus II, it significantly reduced the prolactin surge during the afternoon of proestrus. As in these results obtained in the rat, injections of anti-NKA serum to golden hamsters on diestrus II also significantly decreased the prolactin surge in the afternoon of proestrus. These results suggest a possible physiological role of NKA on prolactin secretion, exerting a stimulatory influence on the release of this hormone.     

The Olfactory Bulbs Enhance Reproductive Hormone Secretion in Male Rats on Long or Short Photoperiod

Unlike seasonally breeding species such as the Syrian hamster, Sprague-Dawley laboratory rats do not normally respond to short photoperiod (6L18D) with reproductive regression. However, removal of the olfactory bulbs (BX) unmasks a photoperiodic response in pre-pubertal rats so that blinding or short photoperiod results in an inhibition of reproductive hormone secretion and/or a delay in pubertal development. This is apparently mediated by pineal melatonin which inhibits gonadotropin and/or prolactin secretion, but the mechanism by which BX facilitates the response to photoperiod is not clear. Experiment I was performed to determine serum levels of reproductive hormones at frequent intervals following BX and/or maintenance on short days. Twenty-three-day old male rats were BX or underwent sham BX (SH). They were thereafter maintained on a 14L:10D (long photoperiod) or 6L:18D photoperiod for the duration of the study. At 6 weeks following surgery, BX rats on either photoperiod had smaller testes than the SH groups. At week 8, the BX group on 6L:18D had smaller testes than the other three groups. There were transient reductions in serum luteinizing hormone and follicle-stimulating hormone in the BX rats on short photoperiod, but there were prolonged effects of BX decreasing prolactin levels in rats on long or short photoperiod. In SH rats, testosterone was elevated for weeks 6 through 10 of the study, and BX blocked this increase. Experiment II was performed to determine whether BX alters testosterone feedback on gonadotropin secretion. Twenty-three-day old male rats were BX or underwent SH operation and were then returned to a room on 14L10D. Six to 8 weeks later, all animals were castrated and placed on 6L18D or returned to 14L:10D. Eight weeks following castration, the rats were implanted with Silastic capsules containing 0, 10, 20 or 40mm testosterone. The post-castration increase in serum luteinizing hormone and follicle-stimulating hormone was lower in the BX than SH rats. In long photoperiod, serum luteinizing hormone and follicle-stimulating hormone were often lower in the BX rats receiving no testosterone replacement or lower doses of testosterone than in the SH group receiving similar capsules. Maintenance on short photoperiod increased the responsiveness to testosterone so that even the rats receiving low doses of testosterone had very low luteinizing hormone and follicle-stimulating hormone levels whether they were SH or BX. In summary, BX rats on long or short photoperiod had lower serum prolactin and testosterone levels than the comparable SH group and BX inhibited the post-castration increase in gonadotropin secretion. The data therefore suggest that the olfactory bulbs tonically enhance reproductive hormone secretion (especially around the time of pubertal development).     

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

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

Prolactin Induces Tuberoinfundibular Dopaminergic Neurone Differentiation in Snell Dwarf Mice if Administered Beginning at 3 Days of Age

The hypothalamic tuberoinfundibular dopaminergic (TIDA) neurones secrete dopamine, which inhibits prolactin secretion. TIDA neurone numbers are deficient in Ames (df/df) and Snell (dw/dw) dwarf mice, which lack prolactin, growth hormone and thyroid‐stimulating hormone. Prolactin therapy initiated before 21 days maintains normal‐sized TIDA neurone numbers in df/df mice and, when initiated as early as 7 days, maintains the maximum TIDA neurone numbers observed in dw/dw development, which are decreased compared to those in normal mice. The present study investigated the effect of prolactin dose and species on TIDA neurone development. Snell dwarf and normal mice were treated with saline, 5 μg of ovine prolactin (oPRL), 50 μg of oPRL, or 50 μg of recombinant mouse prolactin (rmPRL) beginning at 3 days of age. Brains were analysed at 45 days using catecholamine histofluorescence, and immunohistochemistry for tyrosine hydroxylase or bromodeoxyuridine. Normal mice had greater (P ≤ 0.01) TIDA neurones than dw/dw, regardless of treatment. TIDA neurones in 50 μg oPRL‐treated dw/dw mice were greater (P ≤ 0.05) than those in 5 μg oPRL‐ and rmPRL‐treated dw/dw mice, which were greater (P ≤ 0.01) than those in saline‐treated dw/dw mice. Fifty microgram oPRL‐treated dw/dw mice also had greater (P < 0.01) TIDA neurone numbers than the maximum numbers observed in untreated dw/dw mice development. Among saline, 5 μg oPRL and 50 μg oPRL treatments, but not rmPRL, A14 neurone numbers were higher (P ≤ 0.01) in normal compared to in dw/dw mice. The mechanism of TIDA neurone recruitment was investigated using bromodeoxyuridine (BrdU) treatment at intervals after 21 days. Mice treated with rmPRL, but not oPRL, had increased BrdU incorporation in the periventricular area surrounding the third ventricle and median eminence and in the arcuate nucleus. The data obtained in the present study indicate that oPRL, but not rmPRL, when given at a high enough dose, induces TIDA neurone differentiation in dw/dw mice. This supports neurotrophic effects of prolactin on TIDA neurones in early postnatal development that extends beyond maintenance of the cell population.     

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.     

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

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

Cyclosporine Effects on in vitro Responsiveness of Anterior Pituitary Hormone Release to Dopamine and Thyrotropin-Releasing Hormone in Young Female Rats

Endocrine side effects of the immunosuppressive drug cyclosporine (CyA) include changes in anterior pituitary hormone secretion. The aim of the present study was to examine the effects of CyA on the responsiveness of in situ and ectopic anterior pituitary prolactin (PRL), growth hormone (GH) and luteinizing hormone (LH) release response to dopamine (DA) and thyrotropin-releasing hormone (TRH) treatment in young female rats, and to evaluate the possible PRL participation in these effects. Thirty day old rats were rendered hyperprolactinemic by transplanting an anterior pituitary gland of a littermate donor, under the kidney capsule, and were then injected with CyA or vehicle for 2 or 8 days. Sham-operated rats were used as controls and treated in the same way. CyA treatment prevented the increase in plasma PRL levels which occurred in controls after pituitary grafting. In vitro basal PRL release of in situ pituitaries from either sham-operated and/or pituitary-grafted animals was decreased by CyA treatment at any point studied. Basal in vitro secretion of GH was only decreased in the in situ pituitaries from grafted animals after 2 days of CyA therapy. The presence of an ectopic pituitary lead to an increase in the in vitro basal LH secretion from in situ pituitaries as compared to those from sham-operated rats. Basal LH release rates were not changed by CyA treatment, although the LH release in vitro did increase in the in situ pituitaries from sham-operated animals treated with the drug for 2 days. DA addition to the incubation media decreased the in vitro release of PRL, GH and LH from the in situ pituitaries of sham-operated and pituitary-grafted animals treated with vehicle. In CyA treated animals, DA decreased in vitro PRL release from the in situ pituitaries of animals, independently of the presence or absence of an ectopic pituitary. Reductions of the in vitro GH and LH release after DA treatment were higher in the in situ pituitaries from grafted animals on day 8 of CyA or vehicle treatment. TRH increased the in vitro release of the three hormones with differential effects related to the length of the treatment with CyA and/or the presence of an ectopic pituitary. In vitro release of PRL and GH by ectopic pituitaries was inhibited by previous treatment with CyA and this effect was decreased proportional to the duration of the treatment with the drug, while LH secretion was not modified. Addition of DA to the incubation media resulted in a marked reduction of in vitro PRL and GH release, but only at day 8 of vehicle treatment on GH release did DA addition to media further decrease the release of both hormones from ectopic pituitaries from animals treated for 2 or 8 days with the drug, whereas LH secretion was not modified. TRH addition to the incubation media of ectopic pituitaries surprisingly reduced PRL and GH secretion on day 8 of CyA treatment or after surgery. The results of these studies suggest that CyA can act directly at the hypophyseal level modifying pituitary responsiveness to external stimuli. CyA seems to exert its main effects on lactotroph activity while its effects on somatotrophs and gonadotrophs are less.     

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

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