Pulsatile GnRH Release Research Articles

Circulating leptin does not appear to provide a signal for triggering the initiation of puberty in the male rhesus monkey (Macaca mulatta).

The notion that circulating leptin may provide a somatic signal for timing the onset of puberty was examined in the male rhesus monkey. Circulating leptin levels were determined at weekly or biweekly intervals by RIA in intact (N=6) and prepubertally castrated monkeys (N=5) from approximately 18 to 30 months of age. Circulating testosterone (T) and gonadotropin levels were used as indices to identify the onset of the pubertal reaugmentation of pulsatile GnRH release (week 0) in intact and castrated animals, respectively. Time courses of the peripubertal changes in leptin concentrations in individual monkeys were normalized to week 0. In the intact group, mean leptin concentrations at this critical stage of development (week -26 to week +9) were unremarkable, ranging from 1.6+/-0.3 (SEM) to 2.4+/-0.6 ng/ml (P>0.05). In agonadal males, the pubertal onset of GnRH pulse generator activity, as reflected by sustained increments in plasma gonadotropin concentrations, also occurred in the absence of changes in circulating leptin levels (P>0.05). These findings indicate that the timing of the onset of puberty in male monkeys is not triggered by rising circulating leptin concentrations.

Activation of LH receptors expressed in GnRH neurons stimulates cyclic AMP production and inhibits pulsatile neuropeptide release

The GT1-7 cell line of immortalized GnRH neurons has been shown to express receptors for GnRH, LH, and prolactin, as well as a variety of other hormones and transmitters. Treatment of GT1-7 cells with hCG caused a dose-dependent increase in cAMP production, with a rapid increase during the first 15 min and a subsequent decrease that was prevented by pre-treatment with pertussis toxin. Furthermore, the stimulatory effect of cholera toxin on cAMP production was inhibited by hCG in a dose-dependent manner. These data indicate that the LH receptors expressed in GT1-7 cells are coupled to adenylyl cyclase both stimulatory (Gs) and inhibitory (Gi) proteins. In perifused cell cultures, treatment with forskolin and 8-bromo cAMP increased the amplitude of spontaneous GnRH release. However, treatment with nanomolar concentrations of hCG abolished pulsatile GnRH release from both GT1-7 cells and rat hypothalamic cells. The similarity of hCG action on pulsatile GnRH release to that of extracellular Ca2+ depletion and calcium channel antagonists, and its partial resistance to potassium-induced depolarization, suggest that it results from inhibition of plasma-membrane ion channel activity. It is probable that the inhibitory action of hCG on pulsatile GnRH release is responsible for initiation of the suppression of pituitary LH secretion during pregnancy.

Activation of LH receptors expressed in GnRH neurons stimulates cyclic AMP production and inhibits pulsatile neuropeptide release.

The GT1-7 cell line of immortalized GnRH neurons has been shown to express receptors for GnRH, LH, and prolactin, as well as a variety of other hormones and transmitters. Treatment of GT1-7 cells with hCG caused a dose-dependent increase in cAMP production, with a rapid increase during the first 15 min and a subsequent decrease that was prevented by pre-treatment with pertussis toxin. Furthermore, the stimulatory effect of cholera toxin on cAMP production was inhibited by hCG in a dose-dependent manner. These data indicate that the LH receptors expressed in GT1-7 cells are coupled to adenylyl cyclase both stimulatory (Gs) and inhibitory (Gi) proteins. In perifused cell cultures, treatment with forskolin and 8-bromo cAMP increased the amplitude of spontaneous GnRH release. However, treatment with nanomolar concentrations of hCG abolished pulsatile GnRH release from both GT1-7 cells and rat hypothalamic cells. The similarity of hCG action on pulsatile GnRH release to that of extracellular Ca2+ depletion and calcium channel antagonists, and its partial resistance to potassium-induced depolarization, suggest that it results from inhibition of plasma-membrane ion channel activity. It is probable that the inhibitory action of hCG on pulsatile GnRH release is responsible for initiation of the suppression of pituitary LH secretion during pregnancy.

2408 Gap junction does not participate in mechanisms of pulsatile release of gnrh in the rat hypothalamus

2408 Gap junction does not participate in mechanisms of pulsatile release of gnrh in the rat hypothalamus

Novel associations among gonadotropin-releasing hormone neurons.

GnRH is secreted in bursts into the hypophyseal portal vasculature by a small dispersed population of neurons. The means by which the activity of these intrinsically pulsatile cells is coordinated are unknown. This study was initiated as a continuation of our examination of the synaptic input to these cells and their anatomical relationships. Brain tissue from female rhesus monkeys and male and female rats was prepared for the immunocytochemical demonstration of GnRH. At the light microscopic level, GnRH neurons were occasionally found to be in close apposition. Such pairs (or small groups) were randomly distributed throughout the population of GnRH neurons from the diagonal band of Broca through the anterior hypothalamic area in rats and monkeys and in the medial basal hypothalamus in monkeys. The percentage of neurons found in such associations was small (2-7% in rats and 3-15% in monkeys) and was independent of the hormonal condition of the animal. GnRH neurons, either singly or in pairs, were serially sectioned for electron microscopic examination. The sparsity of synaptic input to the cell body that we had reported earlier on the basis of random sampling was confirmed. No soma had more than a dozen synapses, but none totally lacked innervation. The most significant result of serial reconstruction was the discovery of intercellular bridges or passageways between contiguous pairs of GnRH neurons. These were formed by the fusion of processes extending from the two cells or by fusion and opening of passageways in the membranes along regions of contiguity between the two cells. They were found in four of seven pairs of neurons examined in the rat and in four of eight pairs in the monkey. This syncytial arrangement along with GnRH-GnRH synaptic interactions could contribute to the coordination of dispersed influences on these neurons and the propagation of coordinated pulsatile release of GnRH.

Novel associations among gonadotropin-releasing hormone neurons

GnRH is secreted in bursts into the hypophyseal portal vasculature by a small dispersed population of neurons. The means by which the activity of these intrinsically pulsatile cells is coordinated are unknown. This study was initiated as a continuation of our examination of the synaptic input to these cells and their anatomical relationships. Brain tissue from female rhesus monkeys and male and female rats was prepared for the immunocytochemical demonstration of GnRH. At the light microscopic level, GnRH neurons were occasionally found to be in close apposition. Such pairs (or small groups) were randomly distributed throughout the population of GnRH neurons from the diagonal band of Broca through the anterior hypothalamic area in rats and monkeys and in the medial basal hypothalamus in monkeys. The percentage of neurons found in such associations was small (2-7% in rats and 3-15% in monkeys) and was independent of the hormonal condition of the animal. GnRH neurons, either singly or in pairs, were serially sectioned for electron microscopic examination. The sparsity of synaptic input to the cell body that we had reported earlier on the basis of random sampling was confirmed. No soma had more than a dozen synapses, but none totally lacked innervation. The most significant result of serial reconstruction was the discovery of intercellular bridges or passageways between contiguous pairs of GnRH neurons. These were formed by the fusion of processes extending from the two cells or by fusion and opening of passageways in the membranes along regions of contiguity between the two cells. They were found in four of seven pairs of neurons examined in the rat and in four of eight pairs in the monkey. This syncytial arrangement along with GnRH-GnRH synaptic interactions could contribute to the coordination of dispersed influences on these neurons and the propagation of coordinated pulsatile release of GnRH.

Blockade of Singular Follicle-Stimulating Hormone Secretion and Testicular Development in Photostimulated Djungarian Hamsters (Phodopus Sungorus) by a Gonadotropin-Releasing Hormone Antagonist1

Photostimulated male Djungarian hamsters following placement in a long-day photoperiod exhibit a characteristic rise in serum FSH levels that occurs in the absence of a simultaneous rise in LH levels. It is not known whether this singular FSH secretion is dependent upon a differential responsiveness of the gonadotrophs to the pattern of pulsatile GnRH release or is instead driven by a GnRH-independent mechanism. We have assessed the GnRH dependence of this singular FSH secretion by testing the ability of a potent GnRH antagonist (GnRHa: WY-45760) to block FSH and testicular responses to photostimulation. Photoinhibited hamsters were transferred from a short-day (6L:18D) to a long-day photoperiod (16L:8D). Hamsters received two daily injections of a GnRH antagonist or vehicle (VEH). After 0 (short day), 3, 5, 10, 30, or 40 days the hamsters were killed; plasma was assayed for FSH, LH, and testosterone (T), and testes weights were recorded. Testes were sectioned and analyzed for tubular development. In VEH-treated animals, testicular weights increased after photostimulation, reaching mean values of 514 mg by 30 days. Treatment with GnRHa resulted in a significant (p < 0.01) attenuation of testicular growth after 30 days of photostimulation (mean testes weight = 110.1 mg). In VEH-treated hamsters there was a rapid increase in FSH levels after photostimulation that became significant by 5 days and peaked at 10 days. In the GnRHa-treated group, however, these FSH increments were completely blocked at 5 days and significantly reduced at 10 days compared to the values in the corresponding VEH-treated groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Opioidergic modulation of in vitro pulsatile gonadotropin-releasing hormone release from the isolated medial basal hypothalamus of the male guinea pig.

Endogenous opiates exert a tonic restraint on LH release, which is centrally mediated through inhibition of hypothalamic GnRH secretion. The aim of the present study was, using an in vitro superfusion approach, to perform a detailed study of the effects of opioid receptor activation and blockade on the dimensions of pulsatile GnRH release from the isolated medial basal hypothalamus of the adult male guinea pig. The mu-receptor antagonist naloxone at 10(-3) M (n = 12), 10(-6) M (n = 12), and 10(-9) M (n = 12), the opioid agonist morphine sulfate at the same concentrations (n = 14 for each concentration), or morphine and naloxone together at 10(-3) M (n = 14), was added to the medium alternately either during the first or the second of two consecutive 3-h observation periods. Five-minute fractions (750 microliters) were collected for measurement of GnRH by RIA. Morphine suppressed total GnRH output from the medial basal hypothalami (P < or = 0.01 at 10(-3) M and 10(-6) M; P = NS at 10(-9) M), with mean pulse frequency, mean amplitude of all pulses, and mean sum of pulse amplitudes being significantly inhibited at all morphine concentrations, whether pulse analysis (Ultra pulse analysis algorithm) was performed at a two-coefficient of variation (2CV; P < or = 0.05, P < or = 0.01, and P < or = 0.01, respectively) or a 3CV (P < or = 0.05, P < or = 0.05, and P < or = 0.01, respectively) threshold. A different set of experiments (n = 14), with exposure to 10(-3) M morphine during the second of three consecutive 3-h observation periods, indicated that after morphine withdrawal a gradual recovery of GnRH output and pulse frequency to pretreatment values occurred, whereas pulse amplitude remained significantly suppressed during the posttreatment observation period. The inhibitory actions of 10(-3) M morphine were effectively prevented by the presence of 10(-3) M naloxone. Total GnRH output (P < or = 0.05), mean pulse frequency (P < or = 0.05), mean amplitude of all pulses (P < or = 0.05), and mean of the sum of pulse amplitudes (P < or = 0.01) were increased during opioid blockade with 10(-3) M and 10(-9) M naloxone (either 2CV or 3CV threshold for pulse analysis). A similar trend for 10(-6) M naloxone was only apparent after the exclusion of two outliers with unusually high basal GnRH release.(ABSTRACT TRUNCATED AT 400 WORDS)

Postnatal expression of polysialic acid-neural cell adhesion molecule in the hypothalamus of the male rhesus monkey (Macaca mulatta)

Puberty in primates is triggered by a gonad-independent reinitiation of a pulsatile mode of GnRH release. The purpose of the present study was to begin to examine the hypothesis that this neuroendocrine event is the result of structural or plastic changes within the neural network governing the activity of GnRH neurons. Specifically, we sought to determine whether polysialic acid neural cell adhesion molecule (PSA-NCAM), a plasma membrane-associated glycoprotein that has previously been proposed to be a marker for postnatal neuronal plasticity, was expressed within GnRH neuron containing areas of the rhesus monkey hypothalamus. The study employed male monkeys that were castrated prepubertally. Immunocytochemistry of hypothalamic tissue from four animals of pubertal age employing a monoclonal antibody (12F8) specific for PSA-NCAM revealed the presence of PSA-NCAM immunoreactivity within the region of the arcuate nucleus and median eminence of the medial basal hypothalamus (MBH) and in the region of the organum vasculosum of the lamina terminalis of the rostral hypothalamus, two areas in the monkey brain where GnRH neurons are concentrated. As expected, immunostaining for total NCAM using a polyclonal rabbit antibody to mouse total NCAM was uniformly distributed throughout hypothalamic sections containing the MBH. Double staining showed that some, though not all, GnRH cell bodies of the MBH were located within the PSA-NCAM-immunopositive region of the arcuate nucleus and the median eminence. The pattern of PSA-NCAM immunoreactivity in the MBH of three prepubertal monkeys was similar to that seen for the older animals. Western analysis of a membrane extract from the MBH of a monkey of pubertal age, employing antibody 12F8, identified a broad band of staining at the expected molecular weight for this adhesion molecule. A similar, but less intense, immunoreactive band was observed for the preoptic area. In contrast, an immunoblot of a membrane extract of cerebral cortex was only faintly positive for PSA-NCAM. Taken together, the foregoing findings are consistent with the notion that structural changes within the MBH may underlie the pubertal reinitiation of pulsatile GnRH release. Moreover, the presence of PSA-NCAM in the MBH of prepubertal monkeys suggests that the role, if any, of this molecule in the onset of sexual maturation in primates is permissive in nature.

Postnatal expression of polysialic acid-neural cell adhesion molecule in the hypothalamus of the male rhesus monkey (Macaca mulatta).

Puberty in primates is triggered by a gonad-independent reinitiation of a pulsatile mode of GnRH release. The purpose of the present study was to begin to examine the hypothesis that this neuroendocrine event is the result of structural or plastic changes within the neural network governing the activity of GnRH neurons. Specifically, we sought to determine whether polysialic acid neural cell adhesion molecule (PSA-NCAM), a plasma membrane-associated glycoprotein that has previously been proposed to be a marker for postnatal neuronal plasticity, was expressed within GnRH neuron containing areas of the rhesus monkey hypothalamus. The study employed male monkeys that were castrated prepubertally. Immunocytochemistry of hypothalamic tissue from four animals of pubertal age employing a monoclonal antibody (12F8) specific for PSA-NCAM revealed the presence of PSA-NCAM immunoreactivity within the region of the arcuate nucleus and median eminence of the medial basal hypothalamus (MBH) and in the region of the organum vasculosum of the lamina terminalis of the rostral hypothalamus, two areas in the monkey brain where GnRH neurons are concentrated. As expected, immunostaining for total NCAM using a polyclonal rabbit antibody to mouse total NCAM was uniformly distributed throughout hypothalamic sections containing the MBH. Double staining showed that some, though not all, GnRH cell bodies of the MBH were located within the PSA-NCAM-immunopositive region of the arcuate nucleus and the median eminence. The pattern of PSA-NCAM immunoreactivity in the MBH of three prepubertal monkeys was similar to that seen for the older animals. Western analysis of a membrane extract from the MBH of a monkey of pubertal age, employing antibody 12F8, identified a broad band of staining at the expected molecular weight for this adhesion molecule. A similar, but less intense, immunoreactive band was observed for the preoptic area. In contrast, an immunoblot of a membrane extract of cerebral cortex was only faintly positive for PSA-NCAM. Taken together, the foregoing findings are consistent with the notion that structural changes within the MBH may underlie the pubertal reinitiation of pulsatile GnRH release. Moreover, the presence of PSA-NCAM in the MBH of prepubertal monkeys suggests that the role, if any, of this molecule in the onset of sexual maturation in primates is permissive in nature.

Lactational Amenorrhea

There is no doubt that suckling can cause a substantial delay in the resumption of fertility postpartum in all societies regardless of nutritional status. The variability in duration of infertility is directly related to the different patterns of suckling, an uncontrollable variable specific to the mother and child. Resumption of fertility proceeds in two parts, an initial phase of amenorrhea in which ovarian follicle growth is suppressed or attenuated followed by a resumption of menstrual cycles often associated with inadequate luteal function contributing to the reduced fertility of lactating menstruating women. Suckling appears to suppress the normal pattern of pulsatile release of GnRH and hence LH and prevents the normal growth of follicles. The normal positive feedback effect of estrogen on LH release is abolished, and estradiol exerts an enhanced negative feedback effect on both LH and FSH. Thus, while suckling continues, any follicle that starts to develop and secrete estradiol will inhibit further LH release and therefore stop growing. When suckling declines, the pulsatile pattern of LH returns to normal, sensitivity to estrogen negative feedback declines, and follicle growth can continue and ovulation will occur. Opiates do not appear to be mediators of the suckling-induced suppression of GnRH or the release of prolactin in women. There is no clear role for prolactin per se or nutritional status in the suppression of ovarian activity postpartum. Thus, while the suppression of fertility in breast-feeding women plays an extremely important role in limiting populations, the mechanism whereby the suckling stimulus from the nipple causes the disruption in GnRH release from the hypothalamus remains to be elucidated.

Seasonal changes of gonadotropin-releasing hormone secretion in the ewe.

A sustained volley of high-frequency pulses of GnRH secretion is a fundamental step in the sequence of neuroendocrine events leading to ovulation during the breeding season of sheep. In the present study, the pattern of GnRH secretion into pituitary portal blood was examined in ewes during both the breeding and anestrous seasons, with a focus on determining whether the absence of ovulation during the nonbreeding season is associated with the lack of a sustained increase in pulsatile GnRH release. During the breeding season, separate groups (n = 5) of ovary-intact ewes were sampled during the midluteal phase of the estrous cycle and following the withdrawal of progesterone (removal of progesterone implants) to synchronize onset of the follicular phase. During the nonbreeding season, another two groups (n = 5) were sampled either in the absence of hormonal treatments or following withdrawal of progesterone. Pituitary portal and jugular blood for measurement of GnRH and LH, respectively, were sampled every 10 min for 6 h during the breeding season or for 12 h in anestrus. During the breeding season, mean frequency of episodic GnRH release was 1.4 pulses/6 h in luteal-phase ewes; frequency increased to 7.8 pulses/6 h during the follicular phase (following progesterone withdrawal). In marked contrast, GnRH pulse frequency was low (mean less than 1 pulse/6 h) in both groups of anestrous ewes (untreated and following progesterone withdrawal), but GnRH pulse amplitude exceeded that in both luteal and follicular phases of the estrous cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Generation and synchronization of gonadotropin-releasing hormone (GnRH) pulses: intrinsic properties of the GT1-1 GnRH neuronal cell line.

The immortalized neuronal cell line GT1-1 was used to investigate the endogenous pattern of GnRH release. The GT1-1 cell line was derived from a GnRH-secreting tumor in a transgenic mouse induced by genetically targeted expression of the potent simian virus 40 oncogene encoding tumor antigen. Cells attached to coverslips were superfused in Sykes-Moore chambers with Locke's medium, Ca(2+)-free Locke's medium, or Opti-MEM (another defined medium) for 2 hr, and samples were collected at 4-min intervals. Release of GnRH in 17 of 18 superfusion chambers was seen to be pulsatile when data were analyzed by cluster analysis. No significant differences were observed whether only one or both of the coverslips forming the chamber were coated with cells. Pulses exhibited a mean interpulse interval of 25.8 +/- 1.5 min, a mean duration of 18.8 +/- 1.4 min, and a mean amplitude of 150.5 +/- 6.0% above preceding nadir. The removal of Ca2+ from the Locke's medium resulted in the progressive reduction of the amplitude and eventually in the absence of identifiable pulses. Pulses reappeared after the return of Ca2+ to the medium. It is concluded that the GT1-1 cell line secretes GnRH in a rhythmic pattern. These findings suggest that the pulsatile release of GnRH (GnRH pulse generator) may be an intrinsic characteristic of the GnRH neurons. Synchronization of pulsatile release from individual neurons could be mediated via numerous cell-to-cell contacts observed in the cultured cells on coverslips. Synchronization of GnRH release from cells on two physically separated coverslips forming a chamber would appear to be accomplished by a diffusible mediator.

Reproductive dysfunction in amenorrheic athletes and anorexic patients

Strenuous athletic training and anorexia are associated with a high incidence of amenorrhea. While the physiological and/or psychological stressors that contribute to the development of menstrual dysfunction in these individuals appear to differ, they both involve disorders of the reproductive system that originate in the hypothalamus. The available data suggest that both groups experience alterations in the pulsatile release of GnRH, which in turn produces perturbations in pituitary and ovarian function ultimately leading to menstrual dysfunction. In this review, the reproductive and "stress" hormone profiles in amenorrheic athletes and anorexic patients are compared, and the interaction of these hormonal axes in the occurrence of menstrual dysfunction is discussed.

Current concepts of β-endorphin physiology in female reproductive dysfunction

beta-Endorphin has a role in the regulation of the normal menstrual cycle and possibly in the onset of puberty. We have reviewed the evidence pointing to an alteration in this neuropeptide that may contribute to the pathogenesis of various reproductive dysfunctions. Elevated or high levels of beta-endorphin have been associated with exercise-associated amenorrhea, stress-associated amenorrhea, and polycystic ovarian syndrome. Depressed or low levels of beta-endorphin have been associated with PMS and menopause. Alterations in the levels of beta-endorphin may change the pulsatile release of GnRH via noradrenergic and/or dopaminergic pathways. We have primarily focused on beta-endorphin as representative of the endogenous opioid peptides, but other opioid peptides may also contribute to the pathogenesis of various types of reproductive dysfunction. Perhaps it will become possible to characterize and hone our understanding of the function of beta-endorphin and the other substances composing the endogenous opioid peptides. A better understanding of their role in physiological as well as pathophysiological processes may allow for the development of rational approaches to the treatment of specific disorders pertaining to reproduction. Many questions remain unanswered. Among the most relevant are: what is the precise mechanism of action by which beta-endorphin exerts its influence on pulsatile GnRH release? Is there a functional relationship between CNS and peripheral (serum) levels of beta-endorphin? Are the detected changes in beta-endorphin levels merely associated, or are they a cause of a particular disorder? Since it took almost 40 years between the time prostaglandins were first discovered and eventual realization of their clinical application, it may take some time before the beta-endorphin story is complete.

Gonadotropin-Releasing Hormone Secretion during the Postpartum Anestrous Period of the Ewe1

An increase in episodic release of LH is putatively the initial event leading to the onset of postpartum ovarian cyclicity in ewes. This experiment was conducted to determine the relationship between hypothalamic release of GnRH and onset of pulsatile secretion of LH during postpartum anestrus. Control ewes (n = 7) were monitored during the postpartum period to determine when normal estrous cycles resumed. In controls, the mean interval from parturition to the first postpartum estrus as indicated by a rise in serum progesterone greater than 1 ng/mg was 25.8 +/- 0.6 days. Additional ewes (n = 4-5) at 3, 7, 14, and 21 days postpartum (+/- 1 day) were surgically fitted with cannula for collection of hypophyseal-portal blood. Hypophyseal-portal and jugular blood samples were collected over a 6- to 7-h period at 10-min intervals. The number of GnRH pulses/6 h increased (p less than 0.05) from Day 3 postpartum (2.2 +/- 0.5) to Days 7 and 14 (3.6 +/- 0.2 and 3.9 +/- 0.4, respectively). A further increase (p less than 0.05) in GnRH pulse frequency was observed at Day 21 postpartum (6.4 +/- 0.4 pulses/6 h). Changes in pulsatile LH release paralleled changes observed in pulsatile GnRH release over Days 3, 7, 14, and 21 postpartum (0.83 +/- 0.3, 2.8 +/- 0.4, 2.9 +/- 0.6, and 4.0 +/- 1.1 pulses/6 h, respectively). GnRH pulse amplitude was higher at Day 21 than at Days 3, 7, or 14 postpartum. These findings suggest that an increase in the frequency of GnRH release promotes the onset of pulsatile LH release during postpartum anestrus in ewes.

Circulating concentrations of LH and FSH and pituitary responsiveness to GnRH in intact and ovariectomized suckled beef cows in two levels of body condition

ABSTRACTThe effect of body condition on hypothalamic and pituitary function in post-partum suckled beef cows was studied. Thirty-eight Blue-Grey cows were calved at a body condition score of either 2·35 (L) or 2·80 (H), fed to maintain live weight and body condition thereafter and ovariectomized at either 5 or 9 weeks post partum. The H cows had higher LH pulse frequencies before ovariectomy (0·36 v. 0·28 pulses per h; P &lt; 0·05) and 15 days after ovariectomy (0·86 v. 0·72 pulses per h; P &lt; 0·01), indicating a direct effect of body condition on GnRH release from the hypothalamus. Body condition had no effect on the ability of the pituitary to release LH in response to a physiological dose (2·5 u.g) of GnRH in entire or ovariectomized cows at 5 or 9 weeks post partum. The magnitude of the increase in circulating levels of LH following GnRH injection was greater at 9 weeks compared with 5 weeks post partum (2·23 v. 1·73 u.g/1; P &lt; 0·001) in entire cows, but there was no difference between 5 and 9 weeks in ovariectomized cows. Following ovariectomy there was a 2·5-fold increase in LH pulse frequency within 15 days and LH pulse frequency approached 1 pulse per h in all treatments groups. It is concluded that both the hypothalamus and the pituitary are capable of functioning normally by 5 weeks post partum in suckled beef cows, that the pulsatile release of GnRH from the hypothalamus is inhibited, possibly due to a very high sensitivity to oestradiol, and that the sensitivity of the pituitary to the negative feedback effect of oestradiol may decrease with time post partum.

The Prepubertal Hiatus in Gonadotropin Secretion in the Male Rhesus Monkey (Macaca mulatto) Does not Appear to Involve Endogenous Opioid Pep tide Restraint of Hypothalamic Gonadotropin-Releasing Hormone Release*

To examine the possibility that the prepubertal hiatus in gonadotropin secretion in primates is occasioned by an endogenous opioid peptide (EOP)-dependent suppression of pulsatile GnRH release, the ability of an EOP receptor antagonist, naloxone (NAL), to elicit GnRH release was examined indirectly in the rhesus monkey. For this purpose, six castrated male monkeys, aged 18-24 months, first received an intermitten iv infusion of GnRH (0.1 micrograms/min for 3 min every h) to enhance the responsiveness of the gonadotroph to endogenous GnRH. Acute and chronic blockade of EOP receptors with single bolus injections of NAL at three doses (0.2, 2.0, and 10 mg/kg BW) and a continuous infusion of the antagonist (2 mg/h for 36 h), respectively, failed to elicit significant increments in circulating concentrations of mean LH. In addition, changes in plasma LH concentrations during a chronic intermitten iv infusion of NAL (2 mg/kg BW every 6 h for up to 16 days) were unremarkable. Unequivocal discharges of LH, however, were observed in response to small doses of GnRH (0.3 micrograms/monkey) administered iv after all modes of NAL administration. Taken together, these findings fail to provide evidence for the view that in primates, EOPs underlie the hiatus in pulsatile GnRH release, which in these species is responsible for the quiescence of the pituitary-testicular axis during the greater part of prepubertal development.

Pulsatile Release of Gonadotropin-Releasing Hormone from Hypothalamic Explants Is Restrained by Blockade ofN-Methyl-D,L-Aspartate Receptors*

We have shown previously that N-methyl-D,L-aspartate (NMDA) and kainate, two neuroexcitatory amino acids acting through distinct receptors, may induce the release of GnRH from hypothalamic explants. However, that effect could have no physiological significance, since very high concentrations (50 mM) of NMDA and kainate were required. Here, using agents blocking the activation of receptors to neuroexcitatory amino acids, we evaluated their possible physiological involvement in the pulsatile release of GnRH from the hypothalamus of 50-day-old male rats in vitro. In control conditions (10 nM glycine and 1 mM mg2+), the release of GnRH in 7.5-min fractions collected for 2-4 h showed an obvious pulsatile pattern. The mean (+/- 1 SD) interval between pulses, identified by PULSAR program, was 34.3 +/- 11.4 min. The stimulation of GnRH release by NMDA (50 mM) added to the medium for 7.5 min could be blocked reversibly in the presence of MK-801 (100 microM) using medium without glycine or enriched with Mg2+ (2 mM). The endogenous pulses of GnRH secretion were abolished in the presence of MK-801 or using increased Mg2+ concentrations as well as in the absence of glycine. In contrast, pulsatile release of GnRH was not affected in the presence of 6,7-dinitroquinoxaline-2,3-dione (0.1 mM), a selective inhibitor of kainate and quisqualate receptors which suppressed the increase in GnRH release induced by kainate (50 mM) without affecting the response to NMDA. These data indicate that the physiological mechanism of pulsatile GnRH secretion in the hypothalamus may involve endogenous neuroexcitatory factors acting through NMDA-sensitive receptors.

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.