Gonadotropin-releasing Hormone Pulse Generator Activity Research Articles

Raphe glucose-sensing serotonergic neurons stimulate KNDy neurons to enhance LH pulses via 5HT2CR: rat and goat studies

Dysfunction of central serotonergic neurons is known to cause depressive disorders in humans, who often show reproductive and/or glucose metabolism disorders. This study examined whether dorsal raphe (DR) serotonergic neurons sense high glucose availability to upregulate reproductive function via activating hypothalamic arcuate (ARC) kisspeptin neurons (= KNDy neurons), a dominant stimulator of gonadotropin-releasing hormone (GnRH)/gonadotropin pulses, using female rats and goats. RNA-seq and histological analysis revealed that stimulatory serotonin-2C receptor (5HT2CR) was mainly expressed in the KNDy neurons in female rats. The serotonergic reuptake inhibitor administration into the mediobasal hypothalamus (MBH), including the ARC, significantly blocked glucoprivic suppression of luteinizing hormone (LH) pulses and hyperglycemia induced by intravenous 2-deoxy-D-glucose (2DG) administration in female rats. A local infusion of glucose into the DR significantly increased in vivo serotonin release in the MBH and partly restored LH pulses and hyperglycemia in the 2DG-treated female rats. Furthermore, central administration of serotonin or a 5HT2CR agonist immediately evoked GnRH pulse generator activity, and central 5HT2CR antagonism blocked the serotonin-induced facilitation of GnRH pulse generator activity in ovariectomized goats. These results suggest that DR serotonergic neurons sense high glucose availability to reduce gluconeogenesis and upregulate reproductive function by activating GnRH/LH pulse generator activity in mammals.

Dynamics of pulsatile activities of arcuate kisspeptin neurons in aging female mice.

Reproductive senescence is broadly observed across mammalian females, including humans, eventually leading to a loss of fertility. The pulsatile secretion of gonadotropin-releasing hormone (GnRH), which is essential for gonad function, is primarily controlled by kisspeptin neurons in the hypothalamic arcuate nucleus (ARCkiss), the pulse generator of GnRH. The pulsatility of GnRH release, as assessed by the amount of circulating gonadotropin, is markedly reduced in aged animals, suggesting that the malfunctions of ARCkiss may be responsible for reproductive aging and menopause-related disorders. However, the activity dynamics of ARCkiss during the natural transition to reproductive senescence remain unclear. Herein, we introduce chronic in vivo Ca2+ imaging of ARCkiss in female mice by fiber photometry to monitor the synchronous episodes of ARCkiss (SEskiss), a known hallmark of GnRH pulse generator activity, from the fully reproductive to acyclic phase over 1 year. During the reproductive phase, we find that not only the frequency, but also the intensities and waveforms of individual SEskiss, vary depending on the stage of the estrus cycle. During the transition to reproductive senescence, the integrity of SEskiss patterns, including the frequency and waveforms, remains mostly unchanged, whereas the intensities tend to decline. These data illuminate the temporal dynamics of ARCkiss activities in aging female mice. More generally, our findings demonstrate the utility of fiber-photometry-based chronic imaging of neuroendocrine regulators in the brain to characterize aging-associated malfunction.

Continuous acceleration of neural activity of the GnRH pulse generator during chronic peripheral infusion of neurokinin 3 receptor agonist in goats.

Secretion of pulsatile gonadotropin-releasing hormone (GnRH) is essential for reproduction. Kisspeptin neurons in the arcuate nucleus (ARC), which coexpress neurokinin B (NKB) and its receptor (NK3R), are believed to be components of the GnRH pulse generator that regulates pulsatile GnRH secretion. We examined the effects of peripheral infusion of senktide, an NK3R selective agonist, on GnRH pulse generator activity by monitoring multiple unit activity (MUA) in the goat ARC. Previous studies have shown that characteristic increases in MUA (MUA volleys) reflect GnRH pulse generator activity. Senktide was infused intravenously or intravaginally for 2 h while recording MUA. Both infusions significantly increased the MUA volley frequency compared with the control. These results demonstrate that peripherally administered senktide acts centrally to sustainably accelerate the neural activity of the GnRH pulse generator throughout the infusion period. This suggests the possibility of practical applications of NK3R agonists for improving reproductive activity in farm animals.

Definition of the estrogen negative feedback pathway controlling the GnRH pulse generator in female mice

The mechanisms underlying the homeostatic estrogen negative feedback pathway central to mammalian fertility have remained unresolved. Direct measurement of gonadotropin-releasing hormone (GnRH) pulse generator activity in freely behaving mice with GCaMP photometry demonstrated striking estradiol-dependent plasticity in the frequency, duration, amplitude, and profile of pulse generator synchronization events. Mice with Cre-dependent deletion of ESR1 from all kisspeptin neurons exhibited pulse generator activity identical to that of ovariectomized wild-type mice. An in vivo CRISPR-Cas9 approach was used to knockdown ESR1 expression selectively in arcuate nucleus (ARN) kisspeptin neurons. Mice with >80% deletion of ESR1 in ARN kisspeptin neurons exhibited the ovariectomized pattern of GnRH pulse generator activity and high frequency LH pulses but with very low amplitude due to reduced responsiveness of the pituitary. Together, these studies demonstrate that estrogen utilizes ESR1 in ARN kisspeptin neurons to achieve estrogen negative feedback of the GnRH pulse generator in mice.

OR17-2 GABAergic Signalling in the Posterodorsal Medial Amygdala Mediates Psychological Stress-induced Suppression of the GnRH Pulse Generator in Female Mice

Abstract Psychological stress is linked to infertility and reproductive dysfunction by suppressing the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator and pulsatile luteinising hormone (LH) secretion. The posterodorsal subnucleus of the medial amygdala (MePD) is a key upstream regulator of GnRH pulse generator activity and displays increased neuronal activation in response to psychological stressors. The MePD is primarily a GABAergic nucleus with a strong GABAergic projection to hypothalamic reproductive centres, however, their functional significance has not been determined. We hypothesis that MePD GABAergic signalling is a crucial mediator of psychological stress-induced suppression of pulsatile LH secretion. We used a chemogenetic approach to selectively silence MePD GABA neurones during psychological stress to determine the effect on pulsatile LH secretion. Female ovariectomised Vgat-cre mice (n=7) were virally infected to selectively express inhibitory hM4DGi-DREADDs in MePD GABA neurones. Mice were exposed to either predator odour (PO) or restraint stress and given an intraperitoneal injection of either saline or the DREADD chemical activator clozapine-N-oxide (CNO, 5mg/kg). Statistical significance was determined using a one-way ANOVA and post-hoc Tukey test. Data are presented as mean ± SEM and p<0.05 was considered significant. PO exposure significantly increased LH inter-pulse interval (IPI) in the saline group (prestress: 19.05 ± 1.16 min vs. PO: 29.28 ± 3.16 min, p<0.05, n=7) and this effect was blocked by DREADD-mediated inhibition of MePD GABA neurones (prestress: 20.59 ± 1.12 min vs. PO: 21.31 ± 1.76 min, p>0.05, n=7). Restraint stress dramatically increased LH IPI in the saline group (prestress: 17.78 ± 1.02 min vs. restraint: 45.71 ± vs 5.50 min, p<0.05, n=7) and DREADD-mediated inhibition of MePD GABA neurones blocked this effect (prestress: 18.57 ± 1.97 min vs. restraint: 27.26 ± 4.02 min, p>0.05, n=7). Direct projections from the MePD to the GnRH pulse generator in the hypothalamic arcuate nucleus (ARC) have been identified, however, their phenotype is unknown. In this study, we optogenetically stimulated potential MePD GABAergic projection neurone terminals in the ARC and determined the effect on LH IPI. MePD GABA neurones in female Vgat-cre ovariectomised mice (n=6) were virally infected to express channelrhodopsin 2 protein and MePD GABAergic terminals in the ARC were selectively stimulated by blue light via a chronically implanted fibre-optic cannula in the ARC. Sustained optogenetic stimulation at 10 and 20Hz of MePD GABAergic terminals in the ARC dose-dependently suppressed pulsatile LH secretion (LH IPI; control: 23.34 ± 2.11 min vs. 10Hz: 38.08 ± 3.61 min; 20Hz: 56.67 ± 3.33 min, p<0.05, n=6). These findings confirm a functionally significant MePD GABAergic projection to the ARC and highlight the importance of GABA signalling in the MePD in mediating stress-induced suppression of reproductive function. Collectively, these findings provide novel insight into the amygdala neural circuitry underpinning stress-induced reproductive function. Presentation: Sunday, June 12, 2022 11:15 a.m. - 11:30 a.m.

Inverse age-related changes between hypothalamic NPY and KISS1 gene expression during pubertal initiation in male rhesus monkey

The neuroendocrine mechanism underlying the sinusoidal wave nature of gonadotropin–releasing hormone pulse generator activity from infantile to adult age still needs to be meticulously defined. Direct inhibition of kisspeptin neurons by neuropeptide Y (NPY) and close intimacy between the two rekindle the importance of these two neuropeptides controlling reproductive axis activity. Thus, the present study was undertaken to decipher simultaneous fluctuations and to profile correlative changes in the relative expression of KISS1, NPY, and their receptor genes from the mediobasal hypothalamus of infant (n = 3), juvenile, pre-pubertal, and adult (n = 4 in each stage) male rhesus monkey (Macaca mulatta) by RT-qPCR. Significant elevation (p < 0.05−0.01) in KISS1 and KISS1R and low (p < 0.05) expression in NPY and NPY1R mRNA in the adult group as compared to the pre-pubertal group was observed. Moreover, significantly high (p < 0.05) expression of NPY and NPY1R mRNA with non-significant (p> 0.05) decline in KISS1 and KISS1R in pre-pubertal animals in comparison to infants describe inverse correlative age-associated changes during pubertal development. Current findings imply that NPY may contribute as a neurobiological brake for the dormancy of kisspeptin neurons before pubertal onset, while dwindling of this brake is likely to occasion kisspeptin dependent hypothalamic–pituitary–gonadal axis activation at puberty. These findings may help in the development of clinical and therapeutic strategies to regulate fertility in humans.

Local administration of Neurokinin B in the arcuate nucleus accelerates the neural activity of the GnRH pulse generator in goats.

Kisspeptin neurons in the arcuate nucleus (ARC), which co-express neurokinin B (NKB) and dynorphin A, are termed KNDy neurons. These neurons are candidates for the intrinsic gonadotropin-releasing hormone (GnRH) pulse generator. The central and peripheral administration of NKB or its receptor (NK3R) agonist evokes GnRH pulse generator activity and the subsequent pulsatile GnRH/luteinizing hormone (LH) secretion. However, the mechanism responsible for neural activation of the GnRH pulse generator in goats is unclear. We conducted electrophysiological and histochemical experiments to test the hypothesis that KNDy neurons receive NKB and that the signal is transmitted bilaterally to a population of KNDy neurons. Bilateral electrodes aimed at a cluster of KNDy neurons were inserted into the ovariectomized goat ARC. We observed the GnRH pulse generator activity, represented by characteristic increases in multiple-unit activity (MUA volleys). The unilateral administration of NKB or vehicle in the close vicinity of KNDy neurons under simultaneous MUA recording from both sides revealed that only NKB evoked MUA volley(s) immediately after administration. The timing of the MUA volley(s) evoked on the ipsilateral side was synchronized to that on the contralateral side. The double-labeled ISH for KISS1 and TACR3, which encode kisspeptin and NK3R, respectively, revealed that most KNDy neurons co-expressed TACR3. Therefore, NKB could directly stimulate KNDy neurons, following which the stimulatory signal is immediately transmitted to the entire population of KNDy neurons via connection with their fibers. This mechanism helps synchronize burst activity among KNDy neurons, thereby generating neural signals that govern pulsatile GnRH secretion.

Facilitatory and inhibitory role of central amylin administration in the regulation of the gonadotropin-releasing hormone pulse generator activity in goats

Pulsatile gonadotropin-releasing hormone (GnRH) secretion is essential for regulating reproductive functions in mammals. GnRH pulses are governed by a neural mechanism that is termed the GnRH pulse generator. In the present study, we investigated the role of central calcitonin receptor (CTR) signaling in the regulation of the GnRH pulse generator activity in ovariectomized goats by administering amylin, an endogenous ligand for CTR, into the lateral ventricle. GnRH pulse generator activity was measured using multiple unit activity (MUA) recordings in the mediobasal hypothalamus. We analyzed changes in the interval of characteristic increases in MUA (MUA volleys). The MUA volley interval shortened immediately after amylin administration, followed by prolonged intervals. Double in situ hybridization for KISS1 (kisspeptin gene) and CALCR (CTR gene) revealed that low expression levels of CALCR were found in the arcuate kisspeptin neurons, which is suggested as the main population of neurons, involved in GnRH pulse generator activity. These results suggest that central amylin-CTR signaling has a biphasic role in the regulation of GnRH pulse generator activity by acting on cells other than the arcuate kisspeptin neurons in goats.

Peripheral administration of SB223412, a selective neurokinin-3 receptor antagonist, suppresses pulsatile luteinizing hormone secretion by acting on the gonadotropin-releasing hormone pulse generator in estrogen-treated ovariectomized female goats.

Accumulating evidence suggests that KNDy neurons located in the hypothalamic arcuate nucleus (ARC), which are reported to express kisspeptin, neurokinin B, and dynorphin A, are indispensable for the gonadotropin-releasing hormone (GnRH) pulse generation that results in rhythmic GnRH secretion. The aims of the present study were to investigate the effects of peripheral administration of the neurokinin 3 receptor (NK3R/TACR3, a receptor for neurokinin B) antagonist, SB223412, on GnRH pulse-generating activity and pulsatile luteinizing hormone (LH) secretion in ovariectomized Shiba goats treated with luteal phase levels of estrogen. The NK3R antagonist was infused intravenously for 4 h {0.16 or 1.6 mg/(kg body weight [BW]·4 h)} during which multiple unit activity (MUA) in the ARC was recorded, an electrophysiological technique commonly employed to monitor GnRH pulse generator activity. In a separate experiment, the NK3R antagonist (40 or 200 mg/[kg BW·day]) was administered orally for 7 days to determine whether the NK3R antagonist could modulate pulsatile LH secretion when administered via the oral route. Intravenous infusion of the NK3R antagonist significantly increased the interval of episodic bursts of MUA compared with that of the controls. Oral administration of the antagonist for 7 days also significantly prolonged the interpulse interval of LH pulses. The results of this study demonstrate that peripheral administration of an NK3R antagonist suppresses pulsatile LH secretion by acting on the GnRH pulse generator, suggesting that NK3R antagonist administration could be used to modulate reproductive functions in ruminants.

Peripheral administration of κ-opioid receptor antagonist stimulates gonadotropin-releasing hormone pulse generator activity in ovariectomized, estrogen-treated female goats

Pulsatile gonadotropin-releasing hormone (GnRH) secretion is indispensable for reproduction in mammals. Kisspeptin neurons in the hypothalamic arcuate nucleus (ARC), referred to as KNDy neurons because of the coexpression of neurokinin B and dynorphin A, are considered as components of the GnRH pulse generator that produces rhythmic GnRH secretion. The present study aimed to investigate if peripheral administration of PF-4455242, a κ-opioid receptor (KOR, a dynorphin A receptor) antagonist, facilitates pulsatile luteinizing hormone (LH) secretion and GnRH pulse generator activity in estrogen-treated ovariectomized Shiba goats to determine the possibility of using KOR antagonists to artificially control ovarian activities. PF-4455242 was intravenously infused for 4 h (1 or 10 μmol/kg body weight/4 h) or as a single subcutaneous injection (1 or 10 μmol/kg body weight). In a separate experiment, the same KOR antagonist (10 μmol/kg body weight/4 h) was intravenously infused during the recording of multiple unit activity (MUA) in the ARC that reflects the activity of the GnRH pulse generator to test the effects of KOR antagonist administration on GnRH pulse generator activity. Intravenous infusion and single subcutaneous injection of the KOR antagonist significantly increased the frequency of LH pulses compared with controls. Intravenous infusion of KOR antagonist also significantly increased the frequency of episodic bursts in the MUA. The present study demonstrates that peripherally administered KOR antagonist stimulates pulsatile LH secretion by acting on the GnRH pulse generator, and peripheral administration of PF-4455242 can be used to facilitate pulsatile LH secretion, which in turn facilitates ovarian activities in farm animals.

The Effects of Chronic Subcutaneous Administration of an Investigational Kisspeptin Analog, TAK-683, on Gonadotropin-Releasing Hormone Pulse Generator Activity in Goats

The continuous activation of the kisspeptin receptor by its agonists causes the abrogation of kisspeptin signaling, leading to decreased pulsatile luteinizing hormone (LH) secretion. Employing this phenomenon as a tool for probing kisspeptin action, this study aimed to clarify the role of kisspeptin in gonadotropin-releasing hormone (GnRH) pulse generation in goats. We examined the effects of chronic administration of TAK-683, an investigational kisspeptin analog, on LH secretion, GnRH immunostaining, pituitary responses to exogenous GnRH, and GnRH pulse generator activity, reflected by a characteristic increase in multiple-unit activity (MUA volley). An osmotic pump containing TAK-683 was subcutaneously implanted on day 0. TAK-683 treatment dose-dependently suppressed pulsatile LH secretion on day 1. Higher doses of chronic TAK-683 profoundly suppressed pulsatile LH secretion but had little effect on GnRH immunostaining patterns and pituitary responses to GnRH on day 5. In ovariectomized goats, MUA volleys occurred at approximately every 30 min on day -1. On day 5 of chronic TAK-683 administration, pulsatile LH secretion was markedly suppressed, whereas MUA volleys were similar to those observed on day -1. Male pheromones and senktide (neurokinin B receptor agonist) induced an MUA volley but had no effect on LH secretion during chronic TAK-683 administration. The results indicate that the chronic administration of a kisspeptin analog profoundly suppresses pulsatile LH secretion without affecting GnRH content, pituitary function or GnRH pulse generator activity, and they suggest an indispensable role for kisspeptin signaling in the cascade driving GnRH/LH pulses by the GnRH pulse generator.

Effects of intravenous administration of neurokinin receptor subtype-selective agonists on gonadotropin-releasing hormone pulse generator activity and luteinizing hormone secretion in goats.

Recent evidence suggests that neurokinin B (NKB), a member of the neurokinin (tachykinin) peptide family, plays a pivotal role in gonadotropin-releasing hormone (GnRH) pulse generation. Three types of neurokinin receptors (NKRs), NK1R, NK2R and NK3R, are found in the brain. Although NKB preferentially binds to NK3R, other NKRs are possibly also involved in NKB action. The present study examined the effects of intravenous administration of the NKR subtype-selective agonists GR73632 (NK1R), GR64349 (NK2R), and senktide (NK3R) on GnRH pulse generator activity and luteinizing hormone (LH) secretion. Multiple-unit activity (MUA) was monitored in ovariectomized goats (n = 5) implanted with recording electrodes. Characteristic increases in MUA (MUA volleys) were considered GnRH pulse generator activity. Although three NKR agonists dose-dependently induced an MUA volley and an accompanying increase in LH secretion, the efficacy in inducing the volley markedly differed. As little as 10 nmol of senktide induced an MUA volley in all goats, whereas a dose of 1000 nmol was only effective for the NK1R and NK2R agonists in two and four goats, respectively. When the treatment failed to evoke an MUA volley, no apparent change was observed in the MUA or LH secretion. Similar effects of the NK2R and NK3R agonists were observed in the presence of estradiol. The results demonstrated that NK3R plays a predominant role in GnRH pulse generation and suggested that the contributions of NK1R and NK2R to this mechanism may be few, if any, in goats.

Regulation of gonadotropin secretion by monitoring energy availability.

Nutrition is a principal environmental factor influencing fertility in animals. Energy deficit causes amenorrhea, delayed puberty, and suppression of copulatory behaviors by inhibiting gonadal activity. When gonadal activity is impaired by malnutrition, the signals originating from an undernourished state are ultimately conveyed to the gonadotropin-releasing hormone (GnRH) pulse generator, leading to suppressed secretion of GnRH and luteinizing hormone (LH). The mechanism responsible for energetic control of gonadotropin release is believed to involve metabolic signals, sensing mechanisms, and neuroendocrine pathways. The availabilities of blood-borne energy substrates such as glucose, fatty acids, and ketone bodies, which fluctuate in parallel with changes in nutritional status, act as metabolic signals that regulate the GnRH pulse generator activity and GnRH/LH release. As components of the specific sensing system, the ependymocytes lining the cerebroventricular wall in the lower brainstem integrate the information derived from metabolic signals to control gonadotropin release. One of the pathways responsible for the energetic control of gonadal activity consists of noradrenergic neurons from the solitary tract nucleus in the lower brainstem, projecting to the paraventricular nucleus of the hypothalamus. Further studies are needed to elucidate the mechanisms underlying energetic control of reproductive function.

A Population of Kisspeptin/Neurokinin B Neurons in the Arcuate Nucleus May Be the Central Target of the Male Effect Phenomenon in Goats

Exposure of females to a male pheromone accelerates pulsatile gonadotropin-releasing hormone (GnRH) secretion in goats. Recent evidence has suggested that neurons in the arcuate nucleus (ARC) containing kisspeptin and neurokinin B (NKB) play a pivotal role in the control of GnRH secretion. Therefore, we hypothesized that these neurons may be the central target of the male pheromone. To test this hypothesis, we examined whether NKB signaling is involved in the pheromone action, and whether ARC kisspeptin/NKB neurons receive input from the medial nucleus of the amygdala (MeA)—the nucleus suggested to relay pheromone signals. Ovariectomized goats were implanted with a recording electrode aimed at a population of ARC kisspeptin/NKB neurons, and GnRH pulse generator activity, represented by characteristic increases in multiple-unit activity (MUA) volleys, was measured. Pheromone exposure induced an MUA volley and luteinizing hormone (LH) pulse in control animals, whereas the MUA and LH responses to the pheromone were completely suppressed by the treatment with an NKB receptor antagonist. These results indicate that NKB signaling is a prerequisite for pheromone action. In ovariectomized goats, an anterograde tracer was injected into the MeA, and possible connections between the MeA and ARC kisspeptin/NKB neurons were examined. Histochemical observations demonstrated that a subset of ARC kisspeptin/NKB neurons receive efferent projections from the MeA. These results suggest that the male pheromone signal is conveyed via the MeA to ARC kisspeptin neurons, wherein the signal stimulates GnRH pulse generator activity through an NKB signaling-mediated mechanism in goats.

Electrophysiological and Morphological Evidence for Synchronized GnRH Pulse Generator Activity Among Kisspeptin/Neurokinin B/Dynorphin A (KNDy) Neurons in Goats

Neurons in the arcuate nucleus (ARC) that concomitantly express kisspeptin, neurokinin B (NKB) and dynorphin A are termed KNDy neurons and are likely candidates for the intrinsic gonadotropin-releasing hormone (GnRH) pulse generator. Our hypothesis is that KNDy neurons are functionally and anatomically interconnected to generate discrete neural signals that govern pulsatile GnRH secretion. Our goal was to address this hypothesis using electrophysiological and anatomical experiments in goats. Bilateral electrodes targeting KNDy neurons were implanted into ovariectomized goats, and GnRH pulse generator activity, represented by characteristic increases in multiple-unit activity (MUA volleys), was measured. Spontaneous and pheromone- or senktide (an NKB receptor agonist)-induced MUA volleys were simultaneously recorded from both sides of the ARC. An anterograde tracer, biotinylated dextran amine (BDA), was also injected unilaterally into the ARC of castrated male goats, and the distribution of fibers containing both BDA and NKB was examined using dual-labeling histochemistry. The results showed that MUA volleys, regardless of origin (spontaneous or experimentally induced), occur simultaneously between the right and left sides of the ARC. Tract tracing indicated that axons projecting from NKB neurons in the ARC were directly apposed to other NKB neuronal cells located bilaterally in the ARC. These results demonstrate that GnRH pulse generator activity occurs synchronously between both sides of the ARC in goats and that KNDy neurons are bilaterally interconnected in the ARC via NKB-containing fibers. Taken together, the results suggest that KNDy neurons form a neuronal circuit to synchronize burst activity among KNDy neurons and thereby generate discrete neural signals that govern pulsatile GnRH secretion.

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.

Impaired episodic LH secretion in female mice with GFP in GnRH neurons

The ability to assess the activity of gonadotropin-releasing hormone (GnRH) neurons has been greatly enhanced by transgenic animal models with targeted expression of green fluorescent protein (GFP). However, it has yet to be demonstrated that the GnRH system continues to exhibit a full range of normal physiological functions in the presence of such genetic manipulation. Accordingly, we have used repetitive blood sampling via indwelling venous catheters to define LH secretory patterns in normal and transgenic mice. Transgenic females proved to be reproductively competent as defined by fecundity, appropriate cyclic changes in vaginal cytology in intact adult females, and spontaneous LH surges as well as surges in response to steroid or mating stimuli. The expression of c-fos following such steroid treatment and mating in ovariectomized transgenics was similar to the expression previously reported in nontransgenic mice. Likewise, the percentage of retrogradely labeled GnRH neurons was similar to that reported in nontransgenic mice. However, episodic LH secretion, an index of GnRH pulse generator activity, was dramatically compromised in ovariectomized female transgenics compared with C57BL6 controls of both sexes and castrated transgenic males. Taken together, these findings suggest that the GnRH pulse generator is selectively impaired in ovariectomized females in which GnRH neurons express GFP.

Effects of kisspeptin-10 on the electrophysiological manifestation of gonadotropin-releasing hormone pulse generator activity in the female rat.

Kisspeptins are extraordinarily potent in stimulating gonadotropic hormone secretion via an action on the hypothalamic GnRH neural system. Because the physiological frequency of the GnRH pulse generator is a critical component of the control system that governs reproductive processes, the aim of this study was to examine the effect of kisspeptin-10 on pulsatile LH secretion and on the electrophysiological manifestation of GnRH pulse generator activity to determine frequency modulatory effects. Adult Sprague Dawley rats were ovariectomized and chronically implanted with electrodes in the arcuate nucleus to record the characteristic increases in hypothalamic multiunit electrical activity volleys coincident with the initiation of each LH pulse measured in peripheral blood and/or indwelling cardiac catheters for the collection of blood samples (25 microl) every 5 min for 6-7 h for the measurement of LH. Intravenous infusion of kisspeptin-10 (7.5, 35, and 100 nmol) induced a dose-dependent increase in LH secretion. The stimulatory effect of kisspeptin-10 (100 nmol) on LH secretion was blocked by the GnRH antagonist cetrorelix, precluding a singular action on gonadotropes. Unexpectedly, however, the marked increase in LH release in response to kisspeptin-10 (100 nmol) administration was not accompanied by any change in multiunit electrical activity volley frequency. It seem unlikely, therefore, that kisspeptin-10 has an appreciable frequency modulatory effect on GnRH pulse generator activity in the female rat.

Exposure to ram wool stimulates gonadotropin-releasing hormone pulse generator activity in the female goat

In the sheep and goat, exposure of anestrous females to a conspecific male odor enhances reproductive activity. Interestingly, a previous report indicated that male goat hair stimulated pulsatile luteinizing hormone (LH) secretion in the ewe. In the present study, we addressed whether ram wool affects the gonadotropin-releasing hormone (GnRH) pulse generator activity in the female goat. Five ovariectomized (OVX) goats were chronically implanted with recording electrodes in the mediobasal hypothalamus, and manifestations of the GnRH pulse generator were monitored as characteristic increases in multiple-unit activity (MUA volleys). Wool or hair samples were collected from a mature ram, ewe and male goat, and their effects on the MUA volley were examined. The exposure to ram wool induced an MUA volley within 1 min in all five OVX goats, as did the exposure to male goat hair. The ewe wool had no effect on the timing of an MUA volley occurrence. An invariable association of MUA volleys with LH pulses in the peripheral circulation was also confirmed in two OVX goats exposed to ram wool. The present results clearly indicate that exposure to ram wool stimulates pulsatile GnRH/LH release in the female goat. Since exposure to male goat hair enhances pulsatile LH secretion in the ewe, it is likely that very similar, if not identical, molecules are contained in the male-effect pheromone in the sheep and goat.

Activation of melanocortin receptors accelerates the gonadotropin-releasing hormone pulse generator activity in goats

The present study aims to elucidate whether the central melanocortin receptors [melanocortin-3 and -4 receptors (MC3/4-R)] are involved in regulating GnRH pulse generator activity in female goats. The GnRH pulse generator activity was electrophysiologically assessed at the intervals of characteristic increases in multiple-unit activity (MUA volleys) in the mediobasal hypothalamus. In ovariectomized goats, all doses (0.02, 0.2 and 2 nmol) of MT II, an MC3/4-R agonist, injected into the lateral ventricle significantly shortened MUA volley intervals. The duration of the period during which MT II accelerated MUA volleys was positively correlated with the dose of MT II injected. The stimulatory effect of MT II on the GnRH pulse generator activity was attenuated in the presence of estrogen. Intracerebroventricular injection of SHU9119, an MC3/4-R antagonist, significantly prolonged MUA volley intervals at 1 nmol. MT II (0.2 nmol)-induced acceleration of MUA volleys was partially blocked by the antagonism of MC3/4-R with pre-administered SHU9119 (1 nmol). The present findings demonstrate that MC3/4-R are involved in maintaining GnRH pulse generator activity in goats.