Kevin T. O’Byrne

Down‐Regulation of Hypothalamic Kisspeptin and its Receptor, Kiss1r, mRNA Expression is Associated with Stress‐Induced Suppression of Luteinising Hormone Secretion in the Female Rat

Identification of kisspeptin (Kiss1) and its G protein‐coupled receptor 54 (Kiss1r) as an essential component of the hypothalamic‐pituitary‐gonadal (HPG) axis controlling gonadotrophin secretion raises the possibility that kisspeptin‐Kiss1r signalling may play a critical role in the transduction of stress‐induced suppression of reproduction. We examined the effects of: (i) three different stressors, known to suppress pulsatile luteinising hormone (LH) secretion; (ii) corticotrophin‐releasing factor (CRF); and (iii) corticosterone on Kiss1 and Kiss1r expression in key hypothalamic sites regulating gonadotrophin secretion: the medial preoptic area (mPOA) and arcuate nucleus (ARC). Ovariectomised oestrogen‐replaced rats were implanted with i.v., subcutaneous or i.c.v. cannulae. Blood samples were collected at 5‐min intervals for 5–6 h for detection of LH. Quantitative reverse transcriptase‐polymerase chain reaction was used to determine Kiss1 and Kiss1r mRNA levels in brain punches of the mPOA and ARC collected 6 h after restraint, insulin‐induced hypoglycaemia or lipopolysaccharide stress, or after i.c.v. administration of CRF, or acute or chronic subcutaneous administration of corticosterone. We observed down‐regulation of at least one component of the kisspeptin‐Kiss1r signalling system by each of the stress paradigms within the mPOA and ARC. CRF decreased Kiss1 and Kiss1r expression in both the mPOA and ARC. Both acute and chronic stress levels of corticosterone resulted in a concomitant decrease in Kiss1 and an increase in kiss1r mRNA expression in the mPOA and ARC. This differential regulation of Kiss1 and Kiss1r might account for the lack of effect corticosterone has on pulsatile LH secretion. Considering the pivotal role for kisspeptin‐Kiss1r signalling in the control of the HPG axis, these results suggest that the reduced Kiss1‐Kiss1r expression may be a contributing factor in stress‐related suppression of LH secretion.

Phytoestrogens and gonadotropin-releasing hormone pulse generator activity and pituitary luteinizing hormone release in the rat

Phytoestrogens can produce inhibitory effects on gonadotropin secretion in both animals and humans. The aims of this study were 2-fold: 1) to determine in vivo whether genistein and coumestrol act on the GnRH pulse generator to suppress hypothalamic multiunit electrical activity volleys and associated LH pulses and/or on the pituitary to suppress the LH response to GnRH; and 2) to examine the effect of these phytoestrogens on GnRH-induced pituitary LH release in vitro and to determine whether estrogen receptors are involved. Wistar rats were ovariectomized and chronically implanted with recording electrodes and/or indwelling cardiac catheters, and blood samples were taken every 5 min for 7--11 h. Intravenous infusion of coumestrol (1.6-mg bolus followed by 2.4 mg/h for 8.5 h) resulted in a profound inhibition of pulsatile LH secretion, a 50% reduction in the frequency of hypothalamic multiunit electrical activity volleys, and a complete suppression of the LH response to exogenous GnRH. In contrast, both genistein (1.6-mg bolus followed by 2.4 mg/h for 8.5 h) and vehicle were without effect on pulsatile LH secretion. Coumestrol (10(-5) M; over 2 or 4 h) suppressed GnRH-induced pituitary LH release in vitro, an effect blocked by the antiestrogen ICI 182,780. It is concluded that coumestrol acts centrally to reduce the frequency of the hypothalamic GnRH pulse generator. In addition, the inhibitory effects of coumestrol on LH pulses occur at the level of the pituitary by reducing responsiveness to GnRH via an estrogen receptor-mediated process.

GPR54-Dependent Stimulation of Luteinizing Hormone Secretion by Neurokinin B in Prepubertal Rats

Kisspeptin, neurokinin B (NKB) and dynorphin A (Dyn) are coexpressed within KNDy neurons that project from the hypothalamic arcuate nucleus (ARC) to GnRH neurons and numerous other hypothalamic targets. Each of the KNDy neuropeptides has been implicated in regulating pulsatile GnRH/LH secretion. In isolation, kisspeptin is generally known to stimulate, and Dyn to inhibit LH secretion. However, the NKB analog, senktide, has variously been reported to inhibit, stimulate or have no effect on LH secretion. In prepubertal mice, rats and monkeys, senktide stimulates LH secretion. Furthermore, in the monkey this effect is dependent on kisspeptin signaling through its receptor, GPR54. The present study tested the hypotheses that the stimulatory effects of NKB on LH secretion in intact rats are mediated by kisspeptin/GPR54 signaling and are independent of a Dyn tone. To test this, ovarian-intact prepubertal rats were subjected to frequent automated blood sampling before and after intracerebroventricular injections of KNDy neuropeptide analogs. Senktide robustly induced single LH pulses, while neither the GPR54 antagonist, Kp-234, nor the Dyn agonist and antagonist (U50488 and nor-BNI, respectively) had an effect on basal LH levels. However, Kp-234 potently blocked the senktide-induced LH pulses. Modulation of the Dyn tone by U50488 or nor-BNI did not affect the senktide-induced LH pulses. These data demonstrate that the stimulatory effect of NKB on LH secretion in intact female rats is dependent upon kisspeptin/GPR54 signaling, but not on Dyn signaling.

Corticotrophin-Releasing Factor Alters the Timing of Puberty in the Female Rat

Puberty is a developmental process that is dependent upon activation of the hypothalamic gonadotrophin‐releasing hormone (GnRH) pulse generator. It is well established that the stress neuropeptide, corticotrophin‐releasing factor (CRF), has a profound inhibitory action on GnRH pulse generator frequency. Although stress is known to affect the timing of puberty, the role of CRF is unknown. The present study aimed to test the hypothesis that CRF plays a critical role in the timing of puberty. On postnatal day (pnd) 28, female rat pups were chronically implanted with i.c.v. cannulae and received 14 days of administration of either CRF, CRF receptor antagonist (astressin‐B) or artificial cerebrospinal fluid via an osmotic mini‐pump. A separate group of rats served as nonsurgical controls. As a marker of puberty, rats were monitored for vaginal opening and first vaginal oestrus. Levels of CRF, CRF receptor types 1 and 2 (CRF‐R1, CRF‐R2) mRNA expression in micropunches of the medial preoptic area (mPOA), hypothalamic paraventricular nucleus (PVN) and arcuate nucleus (ARC) were determined across pubertal development; brain tissue was collected from a naive group of rats on pnd 14, 32, on the day of vaginal opening, and pnd 77 (Adult). Administration of CRF resulted in a delay in the onset of puberty, whereas astressin‐B advanced pubertal onset. Additionally, CRF and CRF‐R1 mRNA expression was reduced in the mPOA, but not ARC, at puberty. In the PVN, expression of CRF, but not CRF‐R1 mRNA, was reduced at the time of puberty. These data support the hypothesis that CRF signalling may play an important role in modulating the timing of puberty in the rat.

Kisspeptin signaling in the amygdala modulates reproductive hormone secretion

Kisspeptin (encoded by KISS1) is a crucial activator of reproductive function. The role of kisspeptin has been studied extensively within the hypothalamus but little is known about its significance in other areas of the brain. KISS1 and its cognate receptor are expressed in the amygdala, a key limbic brain structure with inhibitory projections to hypothalamic centers involved in gonadotropin secretion. We therefore hypothesized that kisspeptin has effects on neuronal activation and reproductive pathways beyond the hypothalamus and particularly within the amygdala. To test this, we mapped brain neuronal activity (using manganese-enhanced MRI) associated with peripheral kisspeptin administration in rodents. We also investigated functional relevance by measuring the gonadotropin response to direct intra-medial amygdala (MeA) administration of kisspeptin and kisspeptin antagonist. Peripheral kisspeptin administration resulted in a marked decrease in signal intensity in the amygdala compared to vehicle alone. This was associated with an increase in luteinizing hormone (LH) secretion. In addition, intra-MeA administration of kisspeptin resulted in increased LH secretion, while blocking endogenous kisspeptin signaling within the amygdala by administering intra-MeA kisspeptin antagonist decreased both LH secretion and LH pulse frequency. We provide evidence for the first time that neuronal activity within the amygdala is decreased by peripheral kisspeptin administration and that kisspeptin signaling within the amygdala contributes to the modulation of gonadotropin release and pulsatility. Our data suggest that kisspeptin is a ‘master regulator’ of reproductive physiology, integrating limbic circuits with the regulation of gonadotropin-releasing hormone neurons and reproductive hormone secretion.

Kisspeptin in the medial amygdala and sexual behavior in male rats

Highlights • Kisspeptin in the posterodorsal medial amygdala evokes ex-copular erections in rats.• The mechanism is kisspeptin receptor specific.• Kisspeptin induced GnRH and LH release are not implicated.

The Role of GABAergic Signalling in Stress‐Induced Suppression of Gonadotrophin‐Releasing Hormone Pulse Generator Frequency in Female Rats

Stress exerts profound inhibitory effects on reproductive function by suppressing the pulsatile release of gonadotrophin‐releasing hormone (GnRH) and therefore luteinising hormone (LH). This effect is mediated in part via the corticotrophin‐releasing factor (CRF) system, although another potential mechanism is via GABAergic signalling within the medial preoptic area (mPOA) because this has known inhibitory influences on the GnRH pulse generator and shows increased activity during stress. In the present study, we investigated the role of the preoptic endogenous GABAergic system in stress‐induced suppression of the GnRH pulse generator. Ovariectomised oestradiol‐replaced rats were implanted with bilateral and unilateral cannulae targeting toward the mPOA and lateral cerebral ventricle, respectively; blood samples (25 μl) were taken via chronically implanted cardiac catheters every 5 min for 6 h for the measurement of LH pulses. Intra‐mPOA administration of the specific GABAA receptor antagonist, bicuculline (0.2 pmol each side, three times at 20‐min intervals) markedly attenuated the inhibitory effect of lipopolysaccharide (LPS; 25 μg/kg i.v.) but not restraint (1 h) stress on pulsatile LH secretion. By contrast, restraint but not LPS stress‐induced suppression of LH pulse frequency was reversed by application of the selective GABAB receptor antagonist, CGP‐35348, into the mPOA (1.5 nmol each side, three times at 20‐min intervals). However, intra‐mPOA application of either bicuculline or CGP‐35348 attenuated the inhibitory effect of CRF (1 nmol i.c.v.) on the pulsatile LH secretion. These data indicate a pivotal and differential role of endogenous GABAergic signalling in the mPOA with respect to mediating psychological and immunological stress‐induced suppression of the GnRH pulse generator.

The Role of the Bed Nucleus of the Stria Terminalis in Stress-Induced Inhibition of Pulsatile Luteinising Hormone Secretion in the Female Rat

The bed nucleus of the stria terminalis (BNST) occupies a central position in the neural circuitry regulating the hypothalamic‐pituitary‐adrenocortical axis response to stress. The potential role of the BNST in stress‐induced suppression of the gondotrophin‐releasing hormone (GnRH) pulse generator, the central regulator of the reproductive system, was assessed by examining the effects of micro‐infusion of corticotrophin‐releasing factor (CRF) or its antagonist into the BNST on pulsatile luteinising hormone (LH) secretion or stress‐induced inhibition of LH pulses, respectively. Ovariectomised oestrogen‐treated rats were implanted chronically with bilateral cannulae in the dorsolateral BNST and i.v. catheters. CRF (25, 50 or 100 pmol in 200 nl of artificial cerebrospinal fluid) administered bilaterally into the BNST resulted in a dose‐dependent decrease in LH pulse frequency, and induced Fos expression in glutamic acid decarboxylase immunostained neurones in the medial preoptic area. These results suggest that the activation of hypothalamic GABAergic neurones in response to intra‐BNST administration of CRF may be involved in the suppression of LH pulses. Furthermore, administration of CRF antagonist (280 pmol astressin‐B, three times at 20‐min intervals) into the BNST effectively blocked the suppression of pulsatile LH secretion in response to restraint (1 h) but not hypoglycaemic (0.25 U insulin/kg, i.v.) stress. These data suggest that CRF innervation of the dorsolateral BNST plays a key, but differential, role in stress‐induced suppression of the GnRH pulse generator.

Neonatal endotoxin exposure suppresses experimental autoimmune encephalomyelitis through regulating the immune cells responsivity in the central nervous system of adult rats

Early-life exposure to bacterial endotoxin (lipopolysaccharide, LPS) affects the susceptibility to a variety of systemic organic inflammation in adulthood. To determine the long-term effects of neonatal LPS exposure on inflammatory responses in the central nervous system (CNS) in adulthood, we examined the effects on the development of experimental autoimmune encephalomyelitis (EAE) in adult rats as well as the potential regulatory immune mechanisms involved. The results showed that neonatal LPS exposure significantly reduced the morbidity (p<0.01) and severity (p<0.05) of EAE in adult rats, and decreased inflammatory cell infiltration and demyelination in the CNS compared with neonatal saline controls (p<0.05). Neonatal LPS-treated animals showed reduced activation of microglia and astrocytes, as detected by immunocytochemistry, accompanied by down-regulation of the pro-inflammatory cytokines interleukin-17 and interferon-gamma but up-regulation of anti-inflammatory cytokine interleukin-10 in the CNS (p<0.05). At the same time, cerebrum mRNA levels of the transcription factors T-bet and RORgammat were lower in neonatal LPS-compared with saline- treated animals (p<0.05) accompanied with increased STAT-6 and Foxp3 levels in the neonatal LPS-treated group (p<0.05). These findings suggest that early-life exposure to LPS could provide an important neuroprotective effect on the development of EAE in adult rats due to modulation of inflammatory responses in the CNS.

Stress Regulation of Kisspeptin in the Modulation of Reproductive Function

Stressful stimuli abound in modern society and have shaped evolution through altering reproductive development, behavior, and physiology. The recent identification of kisspeptin as an important component of the hypothalamic regulatory circuits involved in reproductive homeostasis sparked a great deal of research interest that subsequently implicated kisspeptin signaling in the relay of metabolic, environmental, and physiological cues to the hypothalamo-pituitary-gonadal axis. However, although it is widely recognized that exposure to stress profoundly impacts on reproductive function, the roles of kisspeptin within the complex mechanisms underlying stress regulation of reproduction remain poorly understood. We and others have recently demonstrated that a variety of experimental stress paradigms downregulate the expression of kisspeptin ligand and receptor within the reproductive brain. Coincidently, these stressors also inhibit gonadotropin secretion and delay pubertal onset-processes that rely on kisspeptin signaling. However, a modest literature is inconsistent with an exclusively suppressive influence of stress on the reproductive axis and suggests that complicated neural interactions and signaling mechanisms translate the stress response into reproductive perturbations. The purpose of this chapter is to review the evidence for a novel role of kisspeptin signaling in the modulation of reproductive function by stress and to broaden the understanding of this timely phenomenon.