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.     

Does cortisol inhibit pulsatile luteinizing hormone secretion at the hypothalamic or pituitary level?

Elevations in glucocorticoids suppress pulsatile LH secretion in sheep, but the neuroendocrine sites and mechanisms of this disruption remain unclear. Here, we conducted two experiments in ovariectomized ewes to determine whether an acute increase in plasma cortisol inhibits pulsatile LH secretion by suppressing GnRH release into pituitary portal blood or by inhibiting pituitary responsiveness to GnRH. First, we sampled pituitary portal and peripheral blood after administration of cortisol to mimic the elevation stimulated by an immune/inflammatory stress. Within 1 h, cortisol inhibited LH pulse amplitude. LH pulse frequency, however, was unaffected. In contrast, cortisol did not suppress either parameter of GnRH secretion. Next, we assessed the effect of cortisol on pituitary responsiveness to exogenous GnRH pulses of fixed amplitude, duration, and frequency. Hourly pulses of GnRH were delivered to ewes in which endogenous GnRH secretion was blocked by estradiol. Cortisol, again, rapidly and robustly suppressed the amplitude of GnRH-induced LH pulses. We conclude that, in the ovariectomized ewe, cortisol suppresses pulsatile LH secretion by inhibiting pituitary responsiveness to GnRH rather than by suppressing hypothalamic GnRH release.     

Endotoxin inhibits pituitary responsiveness to gonadotropin-releasing hormone

Immune/inflammatory challenges powerfully suppress reproductive neuroendocrine activity. This inhibition is generally considered to be centrally mediated via mechanisms that regulate GnRH secretion. The present study provides two lines of evidence that bacterial endotoxin, a commonly used model of immune/inflammatory challenge, also acts to inhibit pituitary responsiveness to GNRH: In the first experiment, pulsatile secretion of GnRH into pituitary portal blood and LH into peripheral blood were monitored in ovariectomized ewes treated with a low dose of endotoxin. Although this treatment only marginally suppressed GnRH pulsatile secretion, it markedly disrupted LH pulsatility. In extreme cases, the low dose of endotoxin blocked LH pulses without inhibiting endogenous GnRH pulses, thereby uncoupling GnRH and LH pulsatile suppression. In the second experiment, we tested the hypothesis that endotoxin inhibits pituitary responsiveness to exogenous GnRH pulses. Hourly pulses of GnRH were delivered to ovariectomized ewes in which endogenous GnRH secretion was blocked. Endotoxin suppressed the amplitude of GnRH-induced LH pulses. Together, these observations support the conclusion that endotoxin inhibits pituitary responsiveness to GNRH:     

Altered neuroendocrine regulation of gonadotropin secretion in women distance runners

We tested the hypothesis that the neuroendocrine control of gonadotropin secretion is altered in certain women distance runners with secondary amenorrhea. To this end, we quantitated the frequency and amplitude of spontaneous pulsatile LH secretion during a 24-h interval in nine such women. The ability of the pituitary gland to release LH normally was assessed by administration of graded bolus doses of GnRH during the subsequent 8 h. Compared to normally menstruating women, six of nine amenorrheic distance runners had a distinct reduction in spontaneous LH pulse frequency, with one, three, six, five, four, or two pulses per 24 h (normal, 8-15 pulses/24 h). This reduction in LH pulse frequency occurred without any significant alterations in plasma concentrations of estradiol and free testosterone or 24-h integrated serum concentrations of LH, FSH, or PRL. Moreover, in long-distance runners, the capacity of the pituitary gland to release LH was normal or accentuated in response to exogenous pulses of GnRH. In the six women athletes with diminished spontaneous LH pulsatility, acute ovarian responsiveness also was normal, since serum estradiol concentrations increased normally in response to the GnRH-induced LH pulses. Although long-distance runners had significantly lower estimated percent body fat compared to control women, specific changes in pulsatile gonadotropin release did not correlate with degree of body leanness. In summary, certain long-distance runners with secondary amenorrhea or severe oligomenorrhea have unambiguously decreased spontaneous LH pulse frequency with intact pituitary responsiveness to GnRH. This neuroendocrine disturbance may be relevant to exercise-associated amenorrhea, since pulsatile LH release is a prerequisite for cyclic ovarian function. We speculate that such alterations in pulsatile LH release in exercising women reflect an adaptive response of the hypothalamic pulse generator controlling the intermittent GnRH signal to the pituitary gland. The basis for amenorrhea in the remaining runners who have normal pulsatile properties of LH release is not known.     

The inhibitory effects of neurokinin B on GnRH pulse generator frequency in the female rat

Neurokinin B (NKB) and its receptor (neurokinin-3 receptor) are coexpressed with kisspeptin and dynorphin A (Dyn) within neurons of the hypothalamic arcuate nucleus, the suggested site of the GnRH pulse generator. It is thought that these neuropeptides interact to regulate gonadotropin secretion. Using the ovariectomized (OVX) and OVX 17β-estradiol-replaced rat models, we have carried out a series of in vivo neuropharmacological and electrophysiological experiments to elucidate the hierarchy between the kisspeptin, NKB, and Dyn signaling systems. Rats were implanted with intracerebroventricular cannulae and cardiac catheters for frequent (every 5 min) automated serial blood sampling. Freely moving rats were bled for 6 h, with intracerebroventricular injections taking place after a 2-h control bleeding period. A further group of OVX rats was implanted with intra-arcuate electrodes for the recording of multiunit activity volleys, which coincide invariably with LH pulses. Intracerebroventricular administration of the selective neurokinin-3 receptor agonist, senktide (100-600 pmol), caused a dose-dependent suppression of LH pulses and multiunit activity volleys. The effects of senktide did not differ between OVX and 17β-estradiol-replaced OVX animals. Pretreatment with a selective Dyn receptor (κ opioid receptor) antagonist, norbinaltorphimine (6.8 nmol), blocked the senktide-induced inhibition of pulsatile LH secretion. Intracerebroventricular injection of senktide did not affect the rise in LH concentrations after administration of kisspeptin (1 nmol), and neither did kisspeptin preclude the senktide-induced suppression of LH pulses. These data show that NKB suppresses the frequency of the GnRH pulse generator in a Dyn/κ opioid receptor-dependent fashion.     

Suppression of mediobasal hypothalamic gonadotropin-releasing hormone and plasma luteinizing hormone pulsatile patterns by phentolamine in ovariectomized rhesus macaques

In gonadectomized animals, pulses of LH are secreted concurrently with pulsatile hypothalamic GnRH and it is hypothesized that pulses of GnRH are either driven or modulated by episodes of catecholamine release. The objective of this study was to determine if the alpha-adrenergic antagonist phentolamine (PHEN) can simultaneously block the release of GnRH and LH in ovariectomized (OVX) rhesus macaques. In Exp 1, simultaneous peripheral blood and mediobasal hypothalamic push-pull perfusion (PPP) samples were collected remotely at 10-min intervals for 24 h via a swivel/tether device in eight conscious, freely moving OVX rhesus monkeys. Phentolamine was continuously infused iv for 6 h at the rate of 4 mg/kg BW.h in five animals and 20 mg/kg BW.h in three animals. Infusion started at 6 h after the commencement of PPP. Sampling of PPP and blood continued for 12 h after the cessation of PHEN infusion. Exp 2 was carried out to determine if PHEN affects pituitary responsiveness to exogenous GnRH under conditions similar to those in Exp 1. Exogenous GnRH (5 micrograms, iv) was injected as a single bolus at 10-h intervals before, during, and after either a saline (4 ml/h for 6 h) infusion or, 3 weeks later, a PHEN infusion (4 mg/kgBW.h for 6 h) in three OVX females. The results of Exp 1 show that pulsatile patterns of hypothalamic GnRH and LH were either dampened or abolished by PHEN infusion. During the recovery period after PHEN infusion, pulse amplitudes of LH were enhanced, but pulse amplitudes of endogenous GnRH did not differ, as compared to those of corresponding LH and GnRH before infusion of PHEN. Data from Exp 2 suggested that the alpha-adrenergic blocking agent had no effect on the pituitary LH response to exogenous GnRH administration. These results directly support the hypothesis that adrenergic neuronal activities are critical for the pulsatile release of hypothalamic GnRH which governs the pulsatile release of LH in gonadectomized animals.     

Electrical activity of the pulse generator of gonadotropin-releasing hormone in 26-month-old female rats

To know whether age-related changes occur in the activity of the pulse generator of gonadotropin-releasing hormone (GnRH), old (26 months) and young (3 months) female rats were examined by recording multiunit activity (MUA) in the median eminence region of the hypothalamus, concurrently with blood samplings through an intra-atrial cannula at 6-min intervals to determine serum luteinizing hormone (LH) concentrations. We have regarded the MUA showing intermittent increases (volleys) at 20-30 min intervals, followed by LH pulses, as the electrical activity of the GnRH pulse generator. We were successful in recording MUA in 18 (26%) of 69 old ovariectomized rats and in 8 (32%) of 25 young ovariectomized rats. The overall mean (+/-SE) of the interval between MUA volleys in old ovariectomized rats was 35.1 +/- 2.0 min (n = 18), which was significantly longer than that of 22.5 +/- 1.5 min (n = 8) in young ovariectomized rats. The mean interval between LH pulses in old ovariectomized rats was 32.2 +/- 3.6 (n = 10), also being significantly longer than that of 23.3 +/- 1.0 (n = 8) in young ovariectomized rats. Further, the LH pulse amplitude in old rats (0. 95 +/- 0.07 ng/ml) was significantly smaller than in young rats (3. 40 +/- 0.36 ng/ml). The present study also confirmed that the increase in serum LH after intravenous injection of 50 ng GnRH was much smaller in old ovariectomized rats. These results show that the electrical activity of the GnRH pulse generator is certainly reduced with age. Taken together with findings suggesting an age-dependent decrease in stimulated transmitter release, attenuation in both frequency and amplitude of GnRH pulses as well as in pituitary responsiveness to GnRH pulses may account for the decreased pulsatile LH secretion observed in aging rats.     

Estradiol and the inhibition of hypothalamic gonadotropin-releasing hormone pulse generator activity in the rhesus monkey.

In mammals, gonadal function is controlled by a hypothalamic signal generator that directs the pulsatile release of gonadotropin-releasing hormone (GnRH) and the consequent pulsatile secretion of luteinizing hormone. In female rhesus monkeys, the electrophysiological correlates of GnRH pulse generator activity are abrupt, rhythmic increases in hypothalamic multiunit activity (MUA volleys), which represent the simultaneous increase in firing rate of individual neurons. MUA volleys are arrested by estradiol, either spontaneously at midcycle or after the administration of the steroid. Multiunit recordings, however, provide only a measure of total neuronal activity, leaving the behavior of the individual cells obscure. This study was conducted to determine the mode of action of estradiol at the level of single neurons associated with the GnRH pulse generator. Twenty-three such single units were identified by cluster analysis of multiunit recordings obtained from a total of six electrodes implanted in the mediobasal hypothalamus of three ovariectomized rhesus monkeys, and their activity was monitored before and after estradiol administration. The bursting of all 23 units was arrested within 4 h of estradiol administration although their baseline activity was maintained. The bursts of most units reappeared at the same time as the MUA volleys, the recovery of some was delayed, and one remained inhibited for the duration of the study (43 days). The results indicate that estradiol does not desynchronize the bursting of single units associated with the GnRH pulse generator but that it inhibits this phenomenon. The site and mechanism of action of estradiol in this regard remain to be determined.     

Duration and frequency of multiunit electrical activity associated with the hypothalamic gonadotropin releasing hormone pulse generator in the rhesus monkey: differential effects of morphine

The effects of morphine on the frequency and duration of the characteristic bursts or 'volleys' of multiunit electrical activity (MUA) associated with pulsatile pituitary luteinizing hormone (LH) secretion were studied in unanesthetized ovariectomized rhesus monkeys bearing bilateral arrays of electrodes implanted in the mediobasal hypothalamus. Morphine administration resulted in a dose-dependent decrease in MUA volley duration and frequency. When morphine was infused at 10 micrograms/kg/h, the inhibiting effect on volley duration was observed without a change in volley frequency. It is concluded that the frequency and duration of hypothalamic MUA volleys associated with pulsatile LH secretion may be independently regulated.     

Inhibitory actions of keoxifene on luteinizing hormone secretion in pituitary gonadotrophs

The effect of keoxifene (LY 156 758) on GnRH-stimulated LH release and its ability to antagonize estrogen actions were investigated in rat anterior pituitary cells. Estrogens exert either stimulatory or inhibitory effects on GnRH-induced LH secretion in rat pituitary cells depending on the incubation time with the steroid. When pituitary cells were treated for 24 h with 10(-9) M estradiol, the LH response to GnRH was clearly enhanced, and this effect was completely inhibited by 300 nM keoxifene. Short term treatment (4 h) of pituitary cells with 10(-9) M estradiol inhibits GnRH-stimulated LH release, and this effect was also blocked by keoxifene in a dose-dependent manner. In the absence of exogenous estrogen the treatment of pituitary cells for 4 h with increasing concentrations of keoxifene reduced the LH response to 10(-9) M GnRH only at very high concentrations (10(-5) M) of the antiestrogen. After treatment for 24 h, the inhibitory effect of keoxifene was evident at concentrations greater than or equal to 10(-8) M, with a reduction of GnRH-induced LH release by up to 60%. The effects of the antiestrogen were also analyzed in a dynamic culture system, in which pituitary cells grown on microcarrier beads were continuously perifused with medium and stimulated with GnRH in a pulsatile fashion. The LH response to a 2 min pulse of 10(-9) M GnRH was reduced in magnitude after 40 min of perifusion with 10(-9) M estradiol. When keoxifene (300 nM) was present at the same time, the LH response was identical to that observed in vehicle-treated cells. At the concentration of 300 nM, keoxifene per se did not change the responsiveness of the pituitary cells to the GnRH stimulus. These findings show that keoxifene is a potent antagonist of both positive and negative estrogen actions in the pituitary gonadotroph. In addition, after short term treatment with high concentrations or after long term treatment, keoxifene itself exerts an inhibitory effect on GnRH-induced LH secretion.     

Corticotropin-releasing factor and gonadotropin-releasing hormone pulse generator activity in the rhesus monkey. Electrophysiological studies

The effect of corticotropin-releasing factor (CRF) on the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator, the central neuronal system governing pulsatile pituitary luteinizing hormone (LH) secretion, was studied electro-physiologically in 6 ovariectomized rhesus monkeys bearing bilateral arrays of recording electrodes implanted in the mediobasal hypothalamus. 'Volleys' of increased multiunit activity (MUA) were recorded for 6-10 h in animals placed in primate chairs. The circulating concentrations of LH and cortisol were determined by radioimmunoassay in blood samples taken every 10 min for 3-4 h prior to the administration of CRF (200 micrograms, i.v.) and for 3-6 h thereafter. CRF resulted in a significant decrease in the frequency of pulse generator activity in 4 of 6 animals, a significant decrease in the duration of MUA volleys and a rise in circulating cortisol levels in all 6 monkeys. Treatment with metyrapone (30 mg/kg, i.m.), an inhibitor of adrenal steroidogenesis that prevented the CRF-induced rise in serum cortisol levels, did not reverse the inhibitory effects of CRF on the frequency or duration of MUA volleys. The opiate antagonist naloxone (0.8 mg/kg, i.v., 10 min prior to CRF followed by 0.8 mg/kg/h infusion) blocked the effects of CRF on MUA volley frequency in 2 of 3 animals, but failed to block the effect of CRF on MUA volley duration, suggesting that endogenous opioids may mediate the action of CRF on pulse generator frequency but not on duration.     

Simultaneous measurement of gonadotropin-releasing hormone, luteinizing hormone, and follicle-stimulating hormone in the orchidectomized rat

In the present study two recently developed techniques have been combined to enable the simultaneous in vivo determination of pulsatile release of GnRH, LH, and FSH in the orchidectomized rat. The first of these techniques involves the implantation of two vascular catheters and collecting serial blood samples through one while simultaneously infusing a replacement blood mixture through the other; consequently, blood samples can be collected for an extended period of time, and detailed plasma LH and FSH release profiles can be established for individual animals. The second technique involves push-pull perfusion of the pituitary gland to determine changes in GnRH concentration as might be perceived by the gonadotropes. For each animal (n = 6), blood (150 microliters) and push-pull perfusate (200 microliters) samples were collected at 5- and 10-min intervals, respectively, for approximately 6 h, and the hormone release profiles were determined by RIA. All of the rats showed a clear pulsatile release pattern for GnRH, LH, and FSH. Moreover, the interpulse interval was remarkably similar for each of these hormones (36.9, 41.5, and 43.5 min, respectively, as determined by PULSAR). The percentage of GnRH pulses associated with a gonadotropin pulse was 72% for LH and 76% for FSH; only 14% of the pulses were silent for both gonadotropins. These results demonstrate that in the orchidectomized rat the pulsatile pattern of GnRH release is reflected in the pulsatile pattern of not only LH but also FSH. They may, therefore, be construed to support the concept that the pulsatile secretion of both gonadotropins is primarily orchestrated by a single hypothalamic releasing hormone. Alternatively, if two separate hypothalamic releasing hormones do indeed exist (LHRH and FSH-releasing hormone), it would appear that in the orchidectomized rat their episodic release is tightly coupled to the same hypothalamic pulse generator.     

Does melatonin alter pituitary responsiveness to gonadotropin-releasing hormone in the ewe?

The diurnal secretion of melatonin from the pineal gland transduces information about day length to the reproductive axis of many seasonal breeders including the ewe. In the sheep the target for melatonin is thought to be neural, such that the hormone acts through the GnRH pulse generator to produce seasonal alterations in the frequency of pulsatile LH secretion. These effects on the pulse generation mechanism take approximately 50 days to become evident. It is possible that melatonin also exerts direct effects at the level of the pituitary gland to alter responsiveness to GnRH. Such effects have been noted in other species. The site of action of melatonin to regulate pulsatile LH secretion was assessed in the ewe by determining whether the animal's endogenous melatonin acutely modifies pituitary responsiveness to sustained pulsatile administration of GnRH. Using an animal model in which endogenous GnRH was blocked, pituitary responsiveness to hourly pulses of exogenous GnRH was assessed under conditions of both high (dark period) and low (light period) melatonin. No evidence for acute effects of melatonin on pituitary response to GnRH was found. In another experiment, the amplitude and frequency of endogenously generated LH pulses in ovariectomized ewes was found not to change during the 24-hour light/dark cycle. These data lead to the conclusion that melatonin does not act at the pituitary gland to produce acute effects on LH secretion. Rather, our findings are consistent with the hypothesis that the action of melatonin, in this short-day breeder is long term, and is directed towards the neural elements of the hypothalamic pulse-generating mechanism.     

Expression of alpha-subunit and luteinizing hormone (LH) beta messenger ribonucleic acid in the rat during lactation and after pup removal: relationship to pituitary gonadotropin-releasing hormone receptors and pulsatile LH secretion

Pituitary GnRH receptor (GnRH-R) levels and LH secretion are suppressed in the lactating rat. To determine if LH synthesis is also inhibited, we have measured LH subunit mRNA levels in the pituitary of lactating rats. We have also examined the temporal relationship among restoration of GnRH-R, LH secretion, and LH synthesis after withdrawing the sensory stimulus of suckling. Pituitary alpha-subunit and LH beta mRNA levels were sharply reduced on day 10 of lactation in both intact and ovariectomized (OVX) animals compared with those in cycling diestrous rats or OVX controls. Removal of the suckling stimulus from OVX animals led to significant increases in alpha-subunit and LH beta mRNA levels by 24 h. Upon removal of the suckling stimulus from intact rats, alpha-subunit mRNA levels were restored by 48 h, but LH beta mRNA levels did not return to diestrous levels until 72 h. Pituitary GnRH-R levels were clearly up-regulated within 1 day after pup removal. Some LH pulses were observed by 48 h, but consistent plasma LH pulses were not detected until 72 h. When pulsatile GnRH was administered during the 24 h after pup removal from intact rats, the regimen of pulsatile GnRH was successful in inducing LH secretion; however, the restoration of pulsatile LH was not accompanied by increases in alpha-subunit and LH beta mRNA levels. The present studies provide further evidence to support the hypothesis that during lactation, the suppression of pituitary gonadotroph function is mainly due to the loss of hypothalamic GnRH secretion. Our data also show that 1) the restoration of GnRH-R alone is not sufficient to activate LH subunit mRNA and LH secretion; 2) the normal restoration of pulsatile LH secretion and increases in LH subunit mRNA are temporally correlated, as increases in LH secretion appear to precede increases in LH subunit mRNA; and 3) the restoration of pituitary LH subunit mRNA levels and pulsatile LH secretion took longer in the intact rat than in the OVX rat, suggesting that ovarian steroids may play a role in the inhibitory effect of lactation.     

Hypothalamic GnRH pulse generator activity during the estradiol-induced LH surge in ovariectomized goats.

Electrophysiological manifestation of the hypothalamic GnRH pulse generator activity was examined during the LH surge induced by estradiol in ovariectomized goats. The characteristic increases in the frequency of multiple unit activity (MUA volley) associated with the pulsatile secretion of LH were recorded using electrodes implanted bilaterally in the medial basal hypothalamus. Estradiol was infused for 16 h at the rate of 3 micrograms/h, to induce an LH surge 10.0-11.5 h later. Regular recurrence of MUA volley was observed throughout the experimental period including the LH surge, but the interval between the MUA volleys became longer (p < 0.01) after the onset of the LH surge as compared with the pretreatment control period (32.9 +/- 2.1 vs. 60.0 +/- 5.1 min). These results suggest that an increased frequency of LH pulses is not a prerequisite for the LH surge in ovariectomized goats given estradiol, and imply that the positive feedback effects of estradiol on LH secretion appear to be exerted through a neuronal mechanism that is intrinsically different from the GnRH pulse generator.     

Interactions between the noradrenergic and opioid peptidergic systems in controlling the electrical activity of luteinizing hormone-releasing hormone pulse generator in ovariectomized rats.

The effects of noradrenergic and opioid peptidergic receptor blockade, either alone or in combination, on the electrical activity of luteinizing hormone-releasing hormone (LHRH) pulse generator were studied in ovariectomized rats fitted with chronically implanted electrode arrays in the medial basal hypothalamus. Both alpha- and beta-adrenergic receptor antagonists, i.e. phenoxybenzamine (5 mg/kg i.v.) and propranolol (5 mg/kg i.v.), respectively, significantly increased the intervals between characteristic increases (volleys) in hypothalamic multiunit activity (MUA), which were associated with the initiation of LH pulses. In contrast to this, an opioid receptor antagonist naloxone (2 mg/kg i.v.) significantly decreased the intervals between the MUA volleys. Naloxone given after the injection of propranolol induced MUA volleys with a latency of a few minutes. However, when given after the injection of phenoxybenzamine, naloxone failed to induce such immediate MUA volleys. These changes in the intervals between the MUA volleys were faithfully reflected by the pulsatile LH secretion. These results suggest that norepinephrine facilitates LHRH pulse generator activity through both alpha- and beta-adrenergic-receptors, and that the action of opioid peptides on it requires an alpha-adrenergic receptor-mediated mechanism.     

Duration of phasic electrical activity of the hypothalamic gonadotropin-releasing hormone pulse generator and dynamics of luteinizing hormone pulses in the rhesus monkey.

The secretion of luteinizing hormone (LH) by the pituitary gland is a pulsatile phenomenon. In the rhesus monkey, each pulse of LH in the peripheral circulation is associated with a characteristic increase in multiunit electrical activity (MUA) recorded from the medial basal hypothalamus. These "volleys" of electrical activity initiate the release of gonadotropin-releasing hormone (GnRH) into the pituitary portal circulation from the terminals of neurosecretory cells. Their duration varies from 1-3 min in normal, adult intact females to 10-25 min in long-term ovariectomized monkeys. A variety of pharmacological interventions also modify volley duration. The purpose of this investigation was to determine the physiological significance of alterations in volley duration. The dynamics of LH pulses in ovariectomized animals were observed in a number of experimental circumstances in which MUA volley duration was reduced from a maximum of 23 min to a minimum of 4 min without significantly altering their frequency. The magnitude of each LH pulse was assessed by calculating the area under the curve delineated by the time course of LH above baseline. In eight experiments, a linear regression of these values on volley duration failed to reveal a significant correlation between MUA volley duration and the magnitude of LH pulses. These results suggest that all of the GnRH secreted per pulse is released at the onset of each MUA volley, the remainder of the increase in electrical activity having no further action on GnRH secretion, although effects on other systems cannot be excluded.     

Control of gonadotropin secretion in the ovine fetus: the effects of a specific gonadotropin-releasing hormone antagonist on pulsatile luteinizing hormone secretion

To demonstrate the dependence of fetal pituitary LH secretion endogenous GnRH, we studied the effects of bolus iv administration of a specific GnRH antagonist analog [GnRH-Ant; (N-acetyl-D-p-chloro-Phe1,2,D-Trp3,D-Arg6,D-Ala10)GnRH] on pulsatile LH release in 10 chronically cannulated ovine fetuses of 104-129 days gestation (term, 147 days). Vehicle alone was given to 13 control fetuses of 107-125 days gestation. Blood samples for LH determination by RIA (NIH LH S16 standard) were taken after injection of either GnRH-Ant (175-300 micrograms dissolved in 1 ml 5% dextrose in water) or vehicle alone for 1.75-5 h. The efficacy of GnRH receptor blockade was then assessed by a bolus iv challenge with 50 micrograms synthetic GnRH. The mean (+/- SEM) observation period per animal was similar for the two groups (3.8 +/- 0.2 h for GnRH-Ant; 3.6 +/- 0.2 h for controls). The frequency of spontaneous pulsatile LH secretion was significantly decreased in the fetuses given GhRH-Ant (2 pulses over 38 h total observation vs. 13 pulses over 47.3 h in control fetuses; P = 0.006). The average interpulse interval was 19.0 h in the GnRH-Ant group compared to 3.6 h in controls. Although the mean pulse amplitude was lower in the GnRH-Ant group (2.8 +/- 1.2 vs. 7.6 +/- 1.1 ng/ml for controls), this difference was not statistically significant (P = 0.065, by one-tailed t test). The mean peak serum LH concentration in response to the GnRH challenge was significantly blunted in the GnRH-Ant group (4.6 +/- 0.8 vs. 20.6 +/- 1.8 ng/ml for controls; P less than 0.001). These results indicate that GnRH-Ant administration causes a virtual cessation of pulsatile LH discharge. As this GnRH-Ant blocks GnRH action at the receptor level, these data demonstrate that pulsatile LH secretion in the ovine fetus is dependent on endogenous GnRH release as early as 104 days gestation.     

Effects of modifying gonadotrophin-releasing hormone input before and after the oestrogen-induced LH surge in ovariectomized ewes with hypothalamo-pituitary disconnection

The patterns of gonadotrophin-releasing hormone (GnRH) input to the pituitary gland that affect the expression of a positive-feedback event by oestrogen on LH secretion were investigated in ovariectomized ewes with hypothalamo-pituitary disconnection (HPD). In experiment 1, ovariectomized HPD ewes were given hourly i.v. pulses of 250 ng GnRH and an i.m. injection of 50 micrograms oestradiol benzoate (OB). The ewes were given a bolus pulse of 2.25 micrograms GnRH 16 h after injection of OB, followed by half-hourly pulses of 250 ng GnRH for 14 h (treatment A). The LH surge response was significantly (P less than 0.05) greater in these ewes compared with that in ewes given a continuous infusion of GnRH (250 ng/h) after the OB injection, followed by a continuous infusion of 500 ng GnRH/h after the bolus pulse of GnRH (treatment B). When no GnRH was administered after the OB injection, except for the bolus pulse of GnRH (treatment C), the surge response was significantly (P less than 0.05) reduced compared with that in treatment A, and was reduced compared with treatment B. These data suggest that GnRH pulses are important in the generation of the OB-induced LH surge, but that a baseline secretory component can prime the pituitary to some extent. In experiment 2, a doubling of the continuous infusion dose of GnRH used in treatment B to 500 ng/h before the bolus pulse of GnRH and to 1 micrograms/h afterwards (treatment D) gave a similar response compared with treatment A, suggesting that if the baseline input of GnRH is of sufficient magnitude, it can overcome the lack of pulsatile input. In experiment 3, halving the GnRH pulse amplitude used in treatment A from 250 to 125 ng (treatment E) did not reduce the LH surge response, implying that when the GnRH input is in a pulsatile mode, the amplitude of GnRH pulses is less important than the pulsatile nature per se. In experiment 4, removal of GnRH input after the bolus pulse of GnRH (treatment F) significantly (P less than 0.05) reduced the surge response compared with when pulses were maintained (treatment A), indicating that GnRH input is still required once the LH surge has been initiated. Collectively, these experiments show that several forms of GnRH delivery, both pulsatile and baseline, can result in the full expression of a positive-feedback response in ovariectomized ewes treated with oestrogen.     

Disruption of GnRH pulses by anti-GnRH serum and phentolamine obliterates pulsatile LH but not FSH secretion in ovariectomized rabbits

It has been hypothesized that the secretion of gonadotropins, i.e. luteinizing hormone (LH) and follicle-stimulating hormone (FSH), is driven by a synchronized neural network ('pulse generator'). This network, regulated in part by alpha-adrenergic activity, ultimately generates bursts of hypothalamic gonadotropin-releasing hormone (GnRH) release. In this study, we used the push-pull (PP) perfusion technique in ovariectomized rabbits to investigate three aspects of the ('GnRH/gonadotropin pulse generator') hypothesis. The objectives were to determine: (1) if plasma LH and FSH pulses occur concomitantly with mediobasal hypothalamic (MBH-) GnRH pulses, (2) changes in the patterns of pulsatile LH and FSH secretion when pulsatile MBH GnRH signals are interrupted by either local immunoneutralization of GnRH or intravenous infusion of the alpha-adrenergic antagonist phentolamine (PHEN, 4 mg/kg BW), and (3) whether third cerebroventricular (3VT-) GnRH patterns reflect neuronal GnRH release from the MBH. We found that while both plasma LH and FSH patterns were pulsatile, MBH GnRH pulses were significantly coupled only with LH pulses (94% coincidence). Both the local immunoneutralization of MBH GnRH pulses and the PHEN-induced suppression of MBH GnRH pulses obliterated the pulsatile secretion of LH, but not FSH. Neither MBH GnRH nor plasma LH or plasma FSH pulses were concurrent with 3VT GnRH pulses. However, the PP perfusion of the 3VT appeared to alter the pulsatile release of MBH GnRH and pituitary LH. The results support the hypothesis that in the absence of ovarian signals, the 'pulse generator' is maintained by tonic alpha-adrenergic input and that a 'cellular unity' of MBH GnRH release (GnRH pulses) drives the gonadotrophs to secrete LH in pulses. In contrast, the pulsatile release of FSH appears to involve additional nonovarian regulatory events to those controlling LH secretion.