Pulsatile Luteinizing Hormone Secretion Research Articles

Involvement of Neurokinin Receptors in the Control of Pulsatile Luteinizing Hormone Secretion in Rats

It has recently been shown that neurokinin B, a tachykinin, is associated with GnRH pulse generation in sheep and goats. The aim of the present study was to clarify the role of tachykinin receptors in the control of LH secretion in rats. To this end, we evaluated the effect of CS-003, an antagonist for all three neurokinin receptors (NK1, NK2 and NK3 receptors), on pulsatile LH secretion in both sexes of rats with different routes of administration. Both oral and third ventricular administration of CS-003 suppressed LH secretion in both sexes of gonadectomized animals. Furthermore, intact male rats with oral administration of CS-003 showed decreased serum testosterone levels, which might be due to suppressed LH secretion. None of the three subtype-specific neurokinin receptor antagonists showed a significant effect on LH secretion in ovariectomized rats when each antagonist was singly administered. The present results suggest that neurokinins play a role in the control of pulsatile GnRH/LH secretion via multiple neurokinin receptors in both male and female rats.

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.

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.

Neurokinin B and Dynorphin A in Kisspeptin Neurons of the Arcuate Nucleus Participate in Generation of Periodic Oscillation of Neural Activity Driving Pulsatile Gonadotropin-Releasing Hormone Secretion in the Goat

Gonadotropin-releasing hormone (GnRH) neurons in the basal forebrain are the final common pathway through which the brain regulates reproduction. GnRH secretion occurs in a pulsatile manner, and indirect evidence suggests the kisspeptin neurons in the arcuate nucleus (ARC) serve as the central pacemaker that drives pulsatile GnRH secretion. The purpose of this study was to investigate the possible coexpression of kisspeptin, neurokinin B (NKB), and dynorphin A (Dyn) in neurons of the ARC of the goat and evaluate their potential roles in generating GnRH pulses. Using double and triple labeling, we confirmed that all three neuropeptides are coexpressed in the same population of neurons. Using electrophysiological techniques to record multiple-unit activity (MUA) in the medial basal hypothalamus, we found that bursts of MUA occurred at regular intervals in ovariectomized animals and that these repetitive bursts (volleys) were invariably associated with discrete pulses of luteinizing hormone (LH) (and by inference GnRH). Moreover, the frequency of MUA volleys was reduced by gonadal steroids, suggesting that the volleys reflect the rhythmic discharge of steroid-sensitive neurons that regulate GnRH secretion. Finally, we observed that central administration of Dyn-inhibit MUA volleys and pulsatile LH secretion, whereas NKB induced MUA volleys. These observations are consistent with the hypothesis that kisspeptin neurons in the ARC drive pulsatile GnRH and LH secretion, and suggest that NKB and Dyn expressed in those neurons are involved in the process of generating the rhythmic discharge of kisspeptin.

Kisspeptin signalling in the hypothalamic arcuate nucleus regulates GnRH pulse generator frequency in the rat.

BackgroundKisspeptin and its G protein-coupled receptor (GPR) 54 are essential for activation of the hypothalamo-pituitary-gonadal axis. In the rat, the kisspeptin neurons critical for gonadotropin secretion are located in the hypothalamic arcuate (ARC) and anteroventral periventricular (AVPV) nuclei. As the ARC is known to be the site of the gonadotropin-releasing hormone (GnRH) pulse generator we explored whether kisspeptin-GPR54 signalling in the ARC regulates GnRH pulses.Methodology/Principal FindingsWe examined the effects of kisspeptin-10 or a selective kisspeptin antagonist administration intra-ARC or intra-medial preoptic area (mPOA), (which includes the AVPV), on pulsatile luteinizing hormone (LH) secretion in the rat. Ovariectomized rats with subcutaneous 17β-estradiol capsules were chronically implanted with bilateral intra-ARC or intra-mPOA cannulae, or intra-cerebroventricular (icv) cannulae and intravenous catheters. Blood samples were collected every 5 min for 5–8 h for LH measurement. After 2 h of control blood sampling, kisspeptin-10 or kisspeptin antagonist was administered via pre-implanted cannulae. Intranuclear administration of kisspeptin-10 resulted in a dose-dependent increase in circulating levels of LH lasting approximately 1 h, before recovering to a normal pulsatile pattern of circulating LH. Both icv and intra-ARC administration of kisspeptin antagonist suppressed LH pulse frequency profoundly. However, intra-mPOA administration of kisspeptin antagonist did not affect pulsatile LH secretion.Conclusions/SignificanceThese data are the first to identify the arcuate nucleus as a key site for kisspeptin modulation of LH pulse frequency, supporting the notion that kisspeptin-GPR54 signalling in this region of the mediobasal hypothalamus is a critical neural component of the hypothalamic GnRH pulse generator.

Melanocortins Mimic the Effects of Leptin to Restore Reproductive Function in Lean Hypogonadotropic Ewes

Background/Aims: Leptin restores gonadotropic function in lean hypogonadotropic animals by an unknown mechanism. We aimed to test the hypothesis that restoration of gonadotropic function is a result of an upregulation of central acetylated melanocortin production. Methods and Results: Lean ovariectomised (OVX) ewes received intracerebroventricular (i.c.v.) infusions of leptin (or vehicle) for 3 days, which upregulated proopiomelanocortin (POMC) mRNA and restored pulsatile luteinizing hormone (LH) secretion. A melanocortin agonist (MTII), but not naloxone treatment, reinstated pulsatile LH secretion in lean OVX ewes. We treated (i.c.v.) lean OVX ewes with leptin (or vehicle) and measured peptide levels and post-translational modification in the arcuate nucleus (ARC). Levels of β-endorphin (β-END) were lower in lean animals, with no effect of leptin treatment. Desacetyl-α-MSH was the predominant form of α-melanocyte-stimulating hormone (α-MSH) in the ARC and levels were similar in all groups. In another group of lean and normal-weight OVX ewes, we measured the different forms of α-MSH in ARC, hypothalamus (ARC-removed) and the preoptic area (POA). Acetylated α-MSH levels were lower in lean animals in the terminal beds of the hypothalamus and POA but not the ARC. Conclusions: Leptin corrects the hypogonadotropic state in the lean condition by upregulation of POMC gene expression, and may increase transport and acetylation of melanocortins to target cells in the brain. Melanocortin treatment restores LH secretion in lean animals.

Food Availability Affects Orexin A/ Hypocretin-1-Induced Inhibition of Pulsatile Luteinizing Hormone Secretion in Female Rats

Orexin A/hypocretin-1 inhibits pulsatile luteinizing hormone (LH) secretion in female rats. In this study, we investigated whether this inhibition was tied to the fasting state, as suggested by our previous study. We first examined whether orexin A inhibited pulsatile LH secretion when food was available ad libitumduring blood sampling. Next, we investigated the effect of intravenous administration of glucose (400 mg/kg) or lactic acid (negative control; 400 mg/kg) on orexin A-induced inhibition of pulsatile LH secretion. We found that orexin A did not affect pulsatile LH secretion in the presence of food, although it increased feeding behavior. Injection of orexin A significantly inhibited pulsatile LH secretion when food was withheld during blood sampling (p < 0.05); this inhibitory effect was rapidly reversed by intravenous injection of glucose but not lactic acid. Because orexin A did not seem to affect pulsatile LH secretion when food was available ad libitum, we speculate that orexin A has an effect on LH secretion when orexin A-induced hunger is accompanied by stress, such as the absence of food. Furthermore, glucose as well as food may act as a satiety factor in gonadotropin-releasing hormone pulse generation.

Hypothalamic Ghrelin Suppresses Pulsatile Secretion of Luteinizing Hormone via β-Endorphin in Ovariectomized Rats

Objectives: Ghrelin, an endogenous growth hormone secretagogue, is a known accelerator of feeding behavior and suppresses pulsatile secretion of luteinizing hormone (LH) in ovariectomized rats. However, the mechanisms underlying this action remain unclear. We examined the effects of naloxone (NAL), a specific opioid antagonist, on the suppression of pulsatile LH secretion by ghrelin to determine whether β-endorphin (β-END) is involved in this suppressive effect. Methods: Ghrelin was administered intracerebroventricularly, and NAL was injected intravenously in ovariectomized rats; then, serum LH concentrations were measured by radioimmunoassay in blood samples drawn every 6 min for 2 h to analyze pulsatile secretion. Results: Administration of ghrelin significantly reduced mean LH concentration and pulse frequency. Coadministration of NAL with ghrelin significantly restored mean LH concentration and pulse frequency. Conclusion: Suppressive effect of intracerebroventricular injection of ghrelin on pulsatile LH secretion was mediated by β-END, suggesting that hypothalamic ghrelin suppressed pulsatile gonadotropin-releasing hormone secretion via β-END in female rats.

Pulsatile Luteinizing Hormone Secretion in Women with Polycystic Ovary Syndrome under Clomiphene Citrate. Differences between Responders and Nonrespondersa , b)

Although administration of Clomiphene Citrate (CC) is an effective therapy for ovulation induction in PCO patients, 15 to 20% of PCO patients do not ovulate following CC treatment. The aim of this study was to compare the endocrine dynamics (LH pulsatile pattern, FSH and steroid hormone changes) in 6 patients who ovulated (PCOS) and 6 PCO patients who did not ovulate in response to CC (PCOR). Blood was collected at 15 min intervals for 4 hr on day 9 of the first menstrual cycle (control cycle) and on day 9 of the following (treatment) cycle. In this cycle all women received 100 mg CC/day from day 5 to day 9. LH was determined in all samples whereas FSH and estradiol were measured every 60 min. No significant differences were observed regarding the age, body mass index, degree of hirsutism, ovarian volumes and basal levels of reproductive hormones between both groups of PCO women. During the control cycle, the LH pulse amplitude and transverse LH mean concentrations in PCOR patients were higher than in PCOR patients whereas the LH pulse frequency was similar in both groups. No difference was found in FSH and estradiol values between both groups. Compared to the control cycle, PCOR patients showed no change in the pulse amplitudes and no difference in LH, FSH and estradiol values following CC treatment. In contrast, during the CC therapy in PCOS patients an increase in the LH amplitude (p less than 0.05), the transverse mean LH concentration (p less than 0.05), FSH and estradiol was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of preoptic injection of glucagon on luteinizing hormone secretion in ovariectomized rats with or without estrogen priming.

Effects of microinjection of glucagon or GIP into the medial preoptic area on luteinizing hormone (LH) and prolactin (PRL) release were examined in unanesthetized ovariectomized rats with or without estrogen priming. Microinjection of glucagon (1.0 microgram) into the medial preoptic area of ovariectomized estrogen-primed rats significantly facilitated the circadian afternoon rise in LH secretion as compared to the hormone values in control animals microinjected with physiological saline. The timing of the afternoon LH rise was not altered by glucagon and the circadian rise of PRL secretion was not altered by glucagon injected in the preoptic area. The injection of GIP did not have any significant effect on either LH or PRL secretion. In ovariectomized estrogen-unprimed rats, on the other hand, glucagon did not affect the pulsatile LH secretion, but it inhibited PRL secretion. GIP did not affect any hormone secretion. The results show that (1) glucagon, as other secretion family peptides such as secretion and PHI, can stimulate the preoptic LH secretory mechanism that undergoes the circadian clock mechanism under the influence of estrogen, and (2) without estrogen priming, glucagon in the preoptic area inhibits PRL secretion.

Basal and pulsatile secretion of human luteinizing hormone--new methods for the analysis of endocrine secretion processes.

Serum concentrations of hormones show typical time-dependent patterns. The diurnal course of the serum concentrations of the luteinizing hormone (LH) was assessed in 35 healthy females monitoring individual concentrations every ten minutes for 24 hours during the follicular phase of their menstrual cycle. Based on these in vivo data, a mathematical procedure was developed for calculation of the momentary hormone secretion rate of an endocrine gland from the time course of the hormone serum concentrations. By means of this procedure, we were able to define the complete 24 hours secretion pattern of LH, including the fact, that basal and pulsatile secretion could be separated from each other in an objective manner. It is possible to calculate the total amount of LH which is secreted per LH-pulse and the basal LH-secretion rate. Two methods were developed to define the time of the onset of an LH-pulse with a preselected probability. Describing the hormonal secretion pattern with a mathematical model provides the basis for therapeutic application of these methods to program implantable hormone-pumps for the therapy of glandular insufficiency.

Arrest of pulsatile luteinizing hormone (LH) secretion during insulin-induced hypoglycemia (IIH): improvement by intrahypothalamic perfusion with glucose.

Insulin-induced hypoglycemia (IIH), as with many other acute stressors, restrains the activity of the reproductive axis, reducing luteinizing hormone (LH) release. In adult ovariectomized, steroid-primed rats, we investigated the effect of IIH and of mediobasal intrahypothalamic perfusion with glucose (200 mg/dl) on pulsatile LH secretion. IIH led to a significant decrease in all pulsatility parameters studied using PC-Pulsar analysis, e.g. pulse amplitude and frequency, maximum and baseline LH levels (p < 0.05 versus control), and LH overall mean release (p < 0.01 versus control). Intrahypothalamic perfusion with glucose normalized LH pulse frequency, improved maximum and baseline levels, and partially ameliorated LH pulse amplitude and overall mean release. Thus, our results show that the glucoprivic cessation of LH release is restored, at least partially, by an adequate glucose supply to the hypothalamus; it is proposed, in view of these and previous results, that different mechanisms in the CNS may be involved in LH suppression observed during IIH.

Effects of ghrelin on Kisspeptin mRNA expression in the hypothalamic medial preoptic area and pulsatile luteinising hormone secretion in the female rat

The orexigenic gut peptide ghrelin negatively modulates the hypothalamic–pituitary–gonadal (HPG) axis. Hyperghrelinaemia results during negative energy balance, a state often associated with delayed puberty and disrupted fertility, whilst exogenous ghrelin suppresses pulsatile luteinising hormone (LH) secretion. The recent identification of kisspeptin (Kiss1) and its G protein-coupled receptor (GPR)54 (Kiss1r) as an essential component of the HPG axis controlling gonadotrophin secretion raises the possibility that kisspeptin-Kiss1r signalling may play a critical role in the transduction of ghrelin-induced suppression of LH. Ovariectomised oestrogen-replaced rats were implanted with intravenous catheters and blood samples collected for detection of LH pulses prior to and after intravenous administration of ghrelin (3 nM/250 μl) or saline (250 μl) during ad libitum feeding or after overnight fasting. Quantitative RT-PCR was used to determine Kiss1 and Kiss1r mRNA levels in brain punches of the key hypothalamic sites regulating gonadotrophin secretion, the medial preoptic area (mPOA) and arcuate nucleus (ARC), collected 6 h following administration of ghrelin. Ghrelin significantly lowered LH pulse frequency in fed rats, an effect significantly enhanced by food deprivation. Fasting, ghrelin or their combination down-regulated Kiss1, without affecting Kiss1r, expression in the mPOA, and affected the expression of neither in the ARC. Considering the pivotal role for kisspeptin signalling in the activation of the HPG axis, the ability of ghrelin to down-regulate Kiss1 expression in mPOA may be a contributing factor in ghrelin-related suppression of pulsatile LH secretion.

Bovine C-terminal octapeptide of RFamide-related peptide-3 suppresses luteinizing hormone (LH) secretion from the pituitary as well as pulsatile LH secretion in bovines

Gonadotropin-inhibiting hormone (GnIH), observed in quail as a member of the RFamide neuropeptide family, suppresses luteinizing hormone (LH) secretion from the avian pituitary. Rats and cattle have an active gene of another member of the RFamide neuropeptide family, termed RFamide-related peptide-3 (RFRP-3), although bovine RFRP-3 is different from that of rats in both length and amino-acid sequence. A single injection of GnIH or RFRP-3 inhibited LH secretion in rodents, which continued for various periods. This study was conducted to evaluate the effects of bovine C-terminal octapeptide of RFRP-3 (RFRP-3-8) on LH secretion from cultured anterior pituitary (AP) cells of cattle, and the effects of RFRP-3-8 injections on pulsatile LH secretion in castrated male calves. The suppressive effect of RFRP-3-8 on LH secretion from AP cells was observed in the presence of gonadotropin-releasing hormone (GnRH), but not in the absence of GnRH in culture media. In another experiment collecting blood samples serially from castrated male calves with repeated intravenous injections of RFRP-3-8 ( n = 6) or saline ( n = 6), the RFRP-3-8 group showed suppressed LH pulse frequency during the injection period ( P < 0.05); however, the RFRP-3-8 group showed no difference from the saline group in all measures of LH secretion in the postinjection period. In conclusion, our results suggested that RFRP-3-8 suppresses LH secretion from cultured AP cells, as well as LH pulse frequency in cattle.

Responses of gonadotropin secretion to short-term dietary supplementation in ovariectomized goats with different body weights

The responses of luteinizing hormone (LH) and follicle stimulating hormone (FSH) secretion to acute dietary supplementation were studied in goats with different body weights. Ovariectomized Shiba goats ( n = 11) were used and were maintained with a feed of 100% of their energy requirement. They were implanted subcutaneously with an oestradiol capsule and were divided into light (LBW; ≤24 kg, n = 6, mean ± S.D., 21.8 ± 2.7 kg) and heavy body weight (HBW; >24 kg, n = 5, mean ± S.D., 32.0 ± 6.3 kg) groups on the basis of their body weights at 8 days before the start of treatment. At the start of treatment (Day 1), the level of a feed changed to 250% of their energy requirement and this level was maintained for 7 days in both groups. Blood samples were collected daily from Day −7 to Day 7 for the analysis of FSH, glucose, and insulin profiles in plasma. Frequent blood samples were also collected at 10 min intervals for 6 h on Day 0, Day 3, and Day 7 for analysis of LH pulses. LH pulse frequency increased significantly on Day 3 as compared with that on Day 0 in both the HBW (7.4 ± 0.5 pulses/6 h vs. 6.2 ± 0.8 pulses/6 h, p < 0.05) and LBW (6.5 ± 0.8 pulses/6 h vs. 5.5 ± 0.5 pulses/6 h, p < 0.05) groups, whereas it decreased on Day 7 (HBW, 6.4 ± 0.9 pulses/6 h; LBW, 6.3 ± 1.6 pulses/6 h, p > 0.05 vs. Day 0). Plasma glucose and insulin concentrations increased temporarily from Day 2 to Day 4 and then decreased to the level before the start of dietary supplementation in both groups. There was no significant difference in the LH pulse frequency or daily concentrations of FSH, glucose, or insulin between the HBW and LBW groups throughout the experimental period. The present study indicated that acute dietary supplementation stimulates pulsatile LH secretion in parallel with a rise of blood glucose and insulin levels. However, the influence of body weight on these responses between light and heavy animals was not observed.

Twelve-hour pair housing attenuates stress-induced hyperthermia in response to conditioned stimulus

The hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator regulates intermittent GnRH discharge into the pituitary portal circulation, and thereby modulates the pattern of pulsatile secretion of luteinizing hormone (LH). Various internal and external factors first modify the GnRH pulse generator activity, which then alters the pulsatile pattern of gonadotrophin secretion, and eventually influence reproductive function. We have successfully recorded characteristic increases in neuronal activity associated with pulsatile LH secretion from the ovariectomized and cycling female goat by means of a multiple unit activity (MUA) recording technique. The unitary relationship between periodic increases in MUA volleys and LH pulses is well maintained under a variety of experimental conditions, providing evidence that these MUA volleys are the consequence of GnRH pulse generator activity. A procedure that has permitted long-term recording of electrophysiological manifestation of the GnRH pulse generator activity and its research application are described.

A role for the medial preoptic area in CGRP-induced suppression of pulsatile LH secretion in the female rat

Calcitonin gene-related peptide (CGRP) is involved in a variety of stress responses and plays a pivotal role in stress-induced suppression of the GnRH pulse generator in the rat. Intracerebroventricular administration of CGRP suppresses luteinizing hormone (LH) pulses and increases Fos expression within the medial preoptic area (mPOA) and paraventricular nucleus (PVN). The aims of the present study were to investigate whether the mPOA or PVN are sites of action for CGRP-induced suppression of LH pulses and whether lipopolysaccharide (LPS), restraint or insulin-induced hypoglycaemia, stressors known to suppress LH pulses, affect mRNA expression for CGRP and its receptor subunits (calcitonin receptor-like receptor (CL) and RAMP-1) in the mPOA and PVN. Micro-infusion of CGRP (50, 250 or 500 pmol) into the mPOA, but not the PVN, dose-dependently suppressed LH pulse frequency. LPS, restraint and hypoglycaemia suppressed RAMP-1 mRNA, but not CL or CGRP mRNA expression in the mPOA. In the PVN, all three stressors suppressed CL mRNA expression, but only LPS or restraint suppressed RAMP-1 mRNA, and CGRP mRNA was unaffected. These results provide evidence that, unlike the PVN, the mPOA might play an important role in the inhibitory effect of CGRP on pulsatile LH secretion. Additionally, CGRP receptor function may be involved in this brain region in stress-induced suppression of the GnRH pulse generator.

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.

Estradiol Enables Cortisol to Act Directly upon the Pituitary to Suppress Pituitary Responsiveness to GnRH in Sheep

We have shown that cortisol infusion reduced the luteinizing hormone (LH) response to fixed hourly GnRH injections in ovariectomized ewes treated with estradiol during the non-breeding season (pituitary-clamp model). In contrast, cortisol did not affect the response to 2 hourly invariant GnRH injections in hypothalamo-pituitary disconnected ovariectomized ewes during the breeding season. To understand the differing results in these animal models and to determine if cortisol can act directly at the pituitary to suppress responsiveness to GnRH, we investigated the importance of the frequency of GnRH stimulus, the presence of estradiol and stage of the circannual breeding season. In experiment 1, during the non-breeding season, ovariectomized ewes were treated with estradiol, and pulsatile LH secretion was restored with i.v. GnRH injections either hourly or 2 hourly in the presence or absence of exogenous cortisol. Experiments 2 and 3 were conducted in hypothalamo-pituitary disconnected ovariectomized ewes in which GnRH was injected i.v. every 2 h. Experiment 2 was conducted during the non-breeding season and saline or cortisol was infused for 30 h in a cross-over design. Experiment 3 was conducted during the non-breeding and breeding seasons and saline or cortisol was infused for 30 h in the absence and presence of estradiol using a cross-over design. Samples were taken from all animals to measure plasma LH. LH pulse amplitude was reduced by cortisol in the pituitary clamp model with no difference between the hourly and 2-hourly GnRH pulse mode. In the absence of estradiol, there was no effect of cortisol on LH pulse amplitude in GnRH-replaced ovariectomized hypothalamo-pituitary disconnected ewes in either season. The LH pulse amplitude was reduced in both seasons in experiment 3 when cortisol was infused during estradiol treatment. We conclude that the ability of cortisol to reduce LH secretion does not depend upon the frequency of GnRH stimulus and that estradiol enables cortisol to act directly on the pituitary of ovariectomized hypothalamo-pituitary disconnected ewes to suppress the responsiveness to GnRH; this effect occurs in the breeding and non-breeding seasons.

Psychosocial stress suppresses attractivity, proceptivity and pulsatile LH secretion in the ewe

Various stressors suppress pulsatile secretion of luteinizing hormone (LH) in ewes and cortisol has been shown to be a mediator of this effect under various conditions. In contrast, little is known about the impact of stress and cortisol on sexual behavior in the ewe. Therefore, we tested the hypothesis that both psychosocial stress and stress-like levels of cortisol will reduce the level of attractivity, proceptivity and receptivity in addition to suppressing LH secretion in the ewe. In Experiment 1, a layered stress paradigm of psychosocial stress was used, consisting of isolation for 4 h with the addition of restraint, blindfold and noise of a barking dog (predator stress) at hourly intervals. This stress paradigm reduced LH pulse amplitude in ovariectomized ewes. In Experiment 2, ovariectomized ewes were artificially induced into estrus with progesterone and estradiol benzoate treatment and the layered stress paradigm was applied. LH was measured and sexual behavior was assessed using T-mazes and mating tests. Stress reduced pulsatile LH secretion, and also reduced attractivity and proceptivity of ewes but had no effect on receptivity. In Experiment 3, ewes artificially induced into estrus were infused with cortisol for 30 h. Cortisol elevated circulating plasma concentrations of cortisol, delayed the onset of estrus and resulted in increased circling behavior of ewes (i.e. moderate avoidance) during estrus and increased investigation and courtship from rams. There was no effect of cortisol on attractivity, proceptivity or receptivity during estrus. We conclude that psychosocial stress inhibits LH secretion, the ability of ewes to attract rams (attractivity) and the motivation of ewes to seek rams and initiate mating (proceptivity), but cortisol is unlikely to be the principal mediator of these effects.