Effect of leptin on hypothalamic release of GnRH and neurotransmitter amino acids during sexual maturation in female rats.

The purpose of the present study was to analyse the effect of leptin treatment on the hypothalamic release of GnRH, GABA, and the excitatory amino acids (EAA), aspartate (ASP) and glutamate (GLU) involved in NMDA neurotransmission in prepubertal (15 day old) and peripubertal (30 day old) female rats. The animals were treated with a single dose of leptin (30 microg/kg i.p.) and sacrificed 60 min later. Hypothalamic samples were incubated in Earle's medium; GnRH was determined by RIA and GLU, ASP and GABA by HPLC by UV detection. The hypothalamic release of GnRH was increased by leptin at both ages, the release being significantly higher in peripubertal than in prepubertal rats. The levels of hypothalamic GABA release were different in the two groups; whereas in prepubertal rats the hypothalamic release of GABA increased with leptin administration, the neurotransmitter release decreased in the peripubertal group. On the other hand, the release of ASP was modified only in the peripubertal group, where leptin significantly increased its hypothalamic release. No modifications in leptin-induced hypothalamic release of GLU were observed at the two ages studied. In conclusion, the results showed that leptin increased GnRH release by the hypothalamus of prepubertal and peripubertal rats. In peripubertal rats this increase was accompanied by a significant decrease in the hypothalamic release of GABA as well as an enhanced release of ASP. These results and previous reports suggest that at this stage of sexual maturation, leptin exerts an stimulatory effect on GnRH by inducing release of excitatory amino acids (ASP) and reducing release of inhibitory amino acids (GABA) involved in GnRH control. In prepubertal rats the stimulating effect of the adipocyte hormone on GnRH appears to be related to its stimulative action on GABA which at this age increases GnRH release.     

Effect of leptin on LH levels and hypothalamic release of GnRH. Its relationship with the hypothalamic neurotransmitter amino acids system in adult male rats

The objective of the present paper was to determine the effect of leptin on the reproductive axis in adult male rats, as well as the hypothalamic mechanisms involved in this effect. For this purpose, we studied the in vivo effect of leptin in adult male rats on serum LH levels, and the in vitro effect on hypothalamic GnRH and amino acid neurotrasmitter release. For in vivo experiments, animals were injected i.p. with leptin at a dose of 30, 100 and 300 microg/kg. In the in vitro experiments, hypothalamic samples were incubated for 60 min in Earle's medium with leptin: 10(-9), 10(-10) and 10(-12) M for GnRH determination, and 10(-10) M for amino acids evaluation. Finally, we studied the effect of the lowest effective leptin dose on plasma LH levels in peripubertal male rats to compare the effect between this group and adults. Leptin induces significant decreases of serum LH levels with the different studied doses (p < 0.01 vs. control) in adult male rats, while in peripubertal male rats, it induced a significant (p < 0.01 vs. control) increment in serum LH levels. On the other hand, in vitro leptin in adult male rats, significantly decreases GnRH release as well as the hypothalamic release of glutamate (GLU). In contrast, leptin increased the GABA release by this hypothalamus in these animals. These results indicate that leptin has an inhibitory effect on the GnRH-LH axis in adult male rats and this effect appears to be connected with an inhibition of hypothalamic release of GLU (the excitatory amino acid) and a stimulatory effect on GABA release (the inhibitory amino acid). On the other hand, in peripubertal male rats, leptin showed a stimulatory effect.     

Changes in the control of gonadotrophin secretion by neurotransmitters during sexual development in rats.

Gonadotrophin hormone releasing hormone (GnRH) is the primary messenger involved in sexual maturation and the onset of puberty. The activity of these neurons are controlled by several neurotransmitters systems. The onset of puberty implies changes from a prepubertal type of gonadotrophin secretion, characterized by a low activity of GnRH neurons, to an adult pattern of secretion with phasic and synchronous activation of GnRH neurons resulting in an increase in the amplitute and frequency of GnRH pulses. Neurotransmitter systems are involved in these changes of GnRH secretion during the onset of puberty by quantitative and qualitative modifications in the effect on GnRH secretion. Serotonin (5-HT), GABA and catecholamines (CA) have qualitative differences in the effects on GnRH and LH secretion in early prepubertal than in late prepubertal and adult female rats. The administration of 5-hydroxytryptophan a precursor of serotonin (5-HT) which increases 5-HT hypothalamic levels induces GnRH and LH release in early prepubertal female rats, these effects dissapear in late prepubertal stage having an inhibitory action in adult female rats. GABAergic system also stimulates GnRH and LH secretion in early prepubertal female rats and has an inhibitory action on this axis in late prepubertal period and in adult female rats. On the contrary the inhibition of catecholamines synthesis by alpha-methyl-p-tyrosine induced an increase of LH secretion in early prepubertal female rats and inhibitory effect in late prepubertal and adult stage. These effects indicate tha CA has an inhibitory effects on GnRH-LH secretion in early prepubertal female rats changing to an stimulatory action in the late puberty and adult rats. These qualitative modifications were observed only in female rats and are probably connected with the hypothalamic differentiation into a female type of gonadotrophin control. Opiadergic and excitatory amino acid systems have quantitative differences on GnRH-LH secretion during prepubertal and peripubertal and adult stages. Opiates has an high inhibitory tone in early prepubertal rats that is decreasing during sexual maturation to reach puberty. On the contrary EAA increases its stimulatory activity on GnRH-LH secretion during sexual maturation by increasing the hypothalamic release of aspartate and glutamate, the excitatory amino acids involved in GnRH release, and the sensibility of NMDA receptors to these amino acids. In conclusion sexual maturation and the onset of puberty in the female rats involve qualitative and quantitative modifications in the effects of neurotrasmitters system on GnRH secretion.     

Leptin stimulates LH secretion in peripubertal male rats through NMDA receptors

Leptin, a peptide hormone secreted by adipocytes that has been proposed as a metabolic signal in the reproductive system, appears to be linked to the different neuroendocrine processes involved in the onset of puberty. We studied the ontogenic effect of administration of leptin (30 mg/kg i.p.) on serum LH levels during different stages of sexual development (7, 30, and 45 days of age) in male rats and on the hypothalamic content of glutamate (GLU) and gamma-aminobutyric acid (GABA) in 30-day-old rats. Leptin induced a significant increase (p < 0.01) in LH levels in 30 days old rats. This hormone stimulatory effect was accompanied by a significant enhancement (p < 0.01) of the hypothalamic content of glutamate, the hypothalamic excitatory aminoacid involved in N-methyl-D-aspartate (NMDA) neurotransmission. No changes in the LH plasma levels were observed in 7- and 45-day-old male rats treated with leptin. MK 801 (0.1 and 0.3 mg/kg i.p.), an antagonist of NMDA receptors of excitatory amino acid system (EAAs), antagonized the stimulatory effect of leptin on LH secretion and on the hypothalamic content of GLU. These results demonstrate that leptin stimulates the reproductive axis in male rats during a determined period of sexual maturation and that NMDA receptors are involved in thefacilitatory action of leptin on the gonadal axis of male rats during sexual maturation.     

Nitric oxide synthase inhibition prevents leptin induced Gn-RH release in prepubertal and peripubertal female rats.

The aim of the present paper was to study the role of NO as a mediator of leptin action at the hypothalamic level during sexual maturation. First, we analyzed the effect of different leptin concentrations (10 (-13), 10 (-11) and 10 (-9) M) on Gn-RH release from anterior preoptic area and medio basal hypothalamus (APOA-MBH) of prepubertal (15 days old) and peripubertal (30 days old) female rats. Leptin 10 (-13) M was the most effective concentration in releasing Gn-RH in both groups of animals. Since glutamate (GLU) and GABA are involved in the hypothalamic control of Gn-RH neurons and also in the neuroendocrine mechanism of puberty, in a second serie of experiments, we evaluated the effect of a competitive inhibitor of nitric oxide synthase (NOS), N-monomethyl-L-arginine (NMMA) on Gn-RH, GLU and GABA release in response to leptin. Co incubation of APOA-MBH with NMMA 0.5 mM, completely blocked Gn-RH and GLU release induced by leptin 10 (-13) M in prepubertal and peripubertal rats. NMMA also blocked the stimulation of GABA release in prepubertal rats, as well as the inhibition of GABA release induced by leptin in peripubertal rats. It can be proposed that the different effect of NO on GABA release, could be related to ontogenic changes, e.g, maturation of receptors and/or interneuronal connections during sexual development. Present results provide evidence that leptin acts at the hypothalamic level to stimulate NO release, which in turn modifies the release of amino acid neurotransmitters involved in Gn-RH control.     

The ontogeny of pulsatile growth hormone secretion and its temporal relationship to the onset of puberty in the agonadal male rhesus monkey (Macaca mulatta)

The pubertal amplification of GH secretion in primates has been thought to reflect an increase in gonadal steroid hormones due to gonadotropin stimulation induced by hypothalamic GnRH release. Previous studies in agonadal, peripubertal, male rhesus monkeys have estimated the age of GnRH activation (defined as d 0) using analyses of nocturnal, pulsatile LH patterns derived from sequential blood samples. Using samples from these earlier studies, secretory patterns of GH were analyzed using Cluster at approximately 30-d intervals in the youngest prepubertal ages and at approximately 10- to 20-d intervals in the period immediately preceding and following the onset of puberty. Pulse frequency, amplitude, and mean GH increased significantly between early prepubertal ages (up to 30 d before d 0) and the late prepubertal period (between -20 d and d 0). Pulsatile GH activity increased earlier than pulsatile LH secretion in four of five animals. These findings support the conclusion that pulsatile GH secretion increases developmentally in the absence of gonadal steroids. Furthermore, the present observation that the developmental increase in GH secretion occurs earlier than previously reported is consistent with the possibility that GH itself either directly or indirectly participates in the pubertal reinitiation of GnRH pulse generator activity.     

Effects of ovarian steroids on the gonadotropin response to N-methyl-D-aspartate and on hypothalamic excitatory amino acid levels during sexual maturation in female rats.

In order to evaluate the involvement of estrogen-progesterone (EP) in the effects of N-methyl-D-aspartate (NMDA) receptor stimulation on gonadotropin secretion during sexual development in female rats, NMDA (30 mg/kg sc) was administered to 16- and 30-day-old female rats pretreated with EP. NMDA administration induced increases in plasma LH concentration that were 13.6-fold and 94.5-fold higher, respectively, than those found after NMDA alone. The increase of LH levels induced by NMDA was accompanied by a significant enhancement of the content of GnRH in the anterior and preoptic hypothalamic areas and in the medial basal hypothalamus (APOA/MBH). EP potentiated this increase of GnRH induced by NMDA. NMDA increased plasma FSH levels at 16 days of age, and this increase was inhibited by EP treatment. In 30-day-old rats EP induced FSH release in response to NMDA. This release was not observed in rats treated only with NMDA. In 16-day-old rats EP induced an increase in the concentrations of aspartate, glutamate, and glycine in the anterior and preoptic hypothalamic areas and in the medial basal hypothalamus, the excitatory amino acids involved in NMDA neurotransmission. This effect was not observed in rats of 30 days of age. In summary, the present results show that during sexual maturation ovarian steroids potentiated the LH-releasing response to NMDA probably by acting at the hypothalamic level; furthermore, during sexual maturation there are changes in the response to EP of the hypothalamic concentrations of excitatory amino acids. These findings could be related to the neuroendocrine mechanisms regulating the onset of puberty and the sexual cycle in female rats.     

A potential apulsatile mode of GnRH release in the male rhesus monkey (Macaca mulatta).

The hypothalamic component of the reproductive axis in vertebrates is comprised of a pulse generator that stimulates the release of GnRH. Several lines of evidence are in agreement that the activity of this pulse generator is intermittent and results in the pulsatile pattern of GnRH and LH release. During a recent investigation of the re-initiation of LH secretion in the agonadal, prepubertal male monkey, we observed a daytime profile of LH secretion, which suggests an apulsatile mode of GnRH release. The first purpose of this study was to describe this observation of apulsatile LH release during the peripubertal transition. Furthermore, we have explored the dependence of this form of LH secretion on GnRH release. Five male rhesus monkeys (Macaca mulatta) were castrated prepubertally and were treated with an intermittent infusion of GnRH to prematurely sensitize the juvenile pituitary to endogenous GnRH release. Alternate daytime (1100-1800 h) and nighttime (1900-0200 h) assessments of LH release were performed at 10-day intervals throughout the peripubertal transition with samples taken every 12 min. In a second experiment, four agonadal males which demonstrated an apulsatile profile of LH release were maintained on an infusion of physiological saline and were treated with the GnRH antagonist Nal-Glu (i.m., 500 microgram/kg). Circulating levels of LH were determined 22 h after antagonist treatment. In peripubertal animals, circulating levels of LH were similar between morning and evening assessments. However, pulse frequency was significantly lower during the daytime. GnRH antagonist reduced LH levels by 72% and a similar reduction in response to an exogenous GnRH test stimulus occurred. These findings suggest an apulsatile mode of GnRH release.     

Octyl-methoxycinnamate (OMC), an ultraviolet (UV) filter, alters LHRH and amino acid neurotransmitters release from hypothalamus of immature rats

OMC (octyl-methoxycinnamate), is an endocrine disruptor with estrogenic activity, which is used in sunscreen creams as a UV filter. We studied its " IN VITRO" effects on the hypothalamic release of LHRH as well as on the amino acid neurotransmitter system in immature rats of 15 (prepubertal) and 30 (peripubertal) days of age. OMC decreased the LH-RH release significantly in male and female rats of both age. In male rats OMC increased the release of GABA while in the female ones It diminished the excitatory amino acid aspartate (ASP) and Glutamate (GLU) without modifications in the hypothalamic GABA release. These results suggest that during sexual maturation the inhibitory effect of OMC on LH-RH release appears to be related to its action on the inhibitory and excitatory amino acid neurotransmitters in male and female rats.     

Characterization of the effects of clomiphene citrate on reproductive physiology in male rats of various ages

Clomiphene citrate (clomiphene) inhibits reproduction in male rats; however, stimulatory effects have been reported at low doses. Male rats were implanted at 60 (adult), 35 (peripubertal) or 10 (prepubertal) days of age with pellets that delivered 0, 0.05, 0.5 or 5.0 mg clomiphene.kg-1.day-1 and were sacrificed after 7 or 14 days of treatment. Testis weight was unaffected by clomiphene in adult and peripubertal rats, but was reduced by all doses in prepubertal rats. Seminal vesicle and prostate gland weights were decreased to varying degrees by clomiphene in all animals, except seminal vesicle weight in peripubertal rats. Serum LH and testosterone were decreased by most doses in all age groups, whereas pituitary LH was decreased in prepubertal rats only. Pituitary GnRH and testicular LH receptor concentrations were reduced in all treated animals. Serum and pituitary FSH were decreased in pre- and peripubertal rats, whereas testicular FSH receptor concentrations were unaffected by treatment. In summary, 1) reproductive function was compromised by clomiphene and many responses were age-dependent, 2) reductions in gonadotropins suggest that clomiphene decreased their synthesis and/or release, and 3) decreased serum LH and testicular LH receptor concentrations were coupled to reduced testosterone secretion.     

Amino acid neurotransmitter release in the preoptic area of rats during the positive feedback actions of estradiol on LH release.

To investigate the role of amino acid neurotransmitters in the regulation of LH secretion in ovariectomized (ovx) rats with or without estrogen substitution, we measured the release rates of gamma-aminobutyric acid (GABA), taurine, glycine, aspartate, glutamate, homocysteic acid, and also of the neurally inactive amino acids serine and glutamine in push-pull perfusate samples of the preoptic/anterior hypothalamic area (PO/AH) collected at 30-min intervals. To achieve this we had to develop a highly sensitive assay utilizing phenylisothiocyanate prederivatization which was followed by HPLC chromatography. In confirmation of our earlier results we observed again a conspicuous drop of preoptic GABA release prior to and during the time of estrogen-induced LH surge. In addition, the release rates of the excitatory amino acid neurotransmitters aspartate and glutamate in the PO/AH increased during this time. Interestingly, also secretion of taurine and glycine was increased during the LH surge, whereas preoptic release rates of serine and glutamine and of homocysteic acid, the putative endogenous ligand of the so-called N-methyl-D-aspartate receptor, remained unchanged. No such changes of amino acid neurotransmitters release rates were observed in ovx rats. This finding underlines that the changes of amino acid secretion in ovx estrogen-primed rats are likely due to the influence of the steroid rather than due to a diurnal rhythm. We conclude that GnRH neurons are under a tonic inhibitory tone exerted by GABA which is relieved during the time of the estrogen-induced LH surge. During this time, aspartate and glutamate may have additional stimulatory effects on GnRH neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Excitatory amino acid neurotransmission evidence for a role in neuroendocrine regulation.

There is compelling evidence that endogenous excitatory amino acid neurotransmission is an important component of the neuroendocrine transmission line that regulates anterior pituitary-hormone release and, thus, reproduction. Excitatory amino acids (EAAs), such as glutamate and aspartate, are found in large quantities in neuroendocrine tissues such as the hypothalamus, and neurons from a variety of hypothalamic nuclei respond with marked excitation to EAA application. Exogenous EAA administration rapidly increases the release of GnRH, LH, and prolactin secretion in vivo and in vitro. Antagonist studies demonstrate that EAA-receptor activation is involved in a number of reproductive-endocrine events, such as the induction of puberty, seasonal breeding, steroid-induced LH secretion, and the preovulatory surge of LH and prolactin in the female. EAA regulation of these neuroendocrine events appears to be achieved through modulation and regulation of hypothalamic GnRH secretion.     

Gonadal-independent activation of enhanced afternoon luteinizing hormone release during pubertal development in the female rat

In the present study we investigated the extent to which expression of the diurnal pattern of LH secretion in prepubertal female rats is driven by an ovarian-independent neuroendocrine mechanism. To remove gonadal influences, rats were ovariectomized (OVX) during early juvenile, late juvenile, or peripubertal phases of development (20-22, 26-27, and 32-33 days of age, respectively), and blood samples were collected throughout the day (every 30-60 min) 2 or 4 days after surgery. Morning plasma LH levels were relatively low 2 days after ovariectomy (30-120 ng/ml), but rose during the afternoon to reach levels ranging from 180-300 ng/ml. This afternoon elevation was sustained both in late juvenile and peripubertal rats, but not in early juvenile rats. The predominant change observed 4 days after ovariectomy was an overall 3- to 6-fold increase in plasma LH levels, which masked the afternoon elevation. Analysis of plasma LH profiles from individual rats, however, revealed that at each of the three ages studied the peak plasma LH levels occurred in the afternoon, and these were most pronounced in the peripubertal animals. To further clarify the existence of such a diurnal pattern of LH release in the 4-day OVX animals pulsatile LH release profiles were obtained from individual animals, using a 5-min bleeding paradigm, at either 24 days (in the morning) or 36 days of age (in either the morning or the afternoon). LH release was episodic in all of the animals studied, with pulses occurring on average of about once every 30 min. Analysis of the plasma LH profiles using the PULSAR algorithm revealed that the overall mean plasma LH levels of the peripubertal animals was enhanced during the afternoon, compared to that in the morning or to the afternoon LH levels in the early juvenile rats. Mean nadir and mean peak LH levels were also greater. No differences in LH pulse amplitude and only marginal differences in LH pulse frequency were detected among the three groups studied. The enhancement of afternoon LH secretion in the OVX rats could not be attributed to age-related or diurnal changes in adenohypophyseal responsiveness to LHRH. The results suggest that the initiation of enhanced afternoon LH secretion, previously shown to occur in intact female rats during the juvenile-peripubertal transition period, results primarily from the activation of a central neuroendocrine mechanism. Although the ovaries might play a role in the development of this diurnal pattern, it is clear that its activation is ovarian independent.     

Substance P stimulates luteinizing hormone secretion from anterior pituitary cells in culture.

Substance P (SP) is present in the anterior pituitary gland (AP), and its concentration there is regulated by the hormonal status of the animal. The observation that SP is releasable from hemipituitaries in a K(+)-stimulated, Ca(2+)-dependent manner and the demonstration of SP-binding sites in the AP have led to the suggestion that SP participates in a paracrine or autocrine manner in the regulation of AP function. Contradictory reports of the effects of SP on the secretion of AP hormones, particularly LH, led us to address the question of whether SP can act directly on the AP to effect LH secretion. We found that SP (100 nM) can stimulate LH release (300-400% of control values) in short term cultures of AP cells and that this effect varies as a function of the age and sex of the animal. There was no significant effect of SP on the release of LH from AP cells of male and female prepubertal rats (20-30 days). During the peripubertal period (30-35 days), a sharp increase in the response to SP occurred in both sexes. This responsiveness was dose dependent and persisted at all ages studied in AP cells from the female rat. In contrast, the responsiveness of AP cells from male rats that developed during the peripubertal period diminished during maturation and was absent after 60 days of age. When adult female rats were exposed to androgens for 6 weeks in vivo and tested for the ability of SP to stimulate the LH secretion, the response was significantly diminished. These studies support the speculation that SP has a functional role in the secretion of LH.     

GnRH antagonist cetrorelix prevents sexual maturation of peripubertal male rats.

Gonadotropin releasing-hormone (GnRH) analogues contain amino acid substitutions of the native decapeptide. Depending on the substitutions, the analogues have GnRH agonistic or antagonistic properties. GnRH agonists are the established treatment in cases of central precocious puberty caused by premature activation of the hypothalamic GnRH pulse generator. Much less data exist on the use of GnRH antagonists to influence the onset of puberty. Using the GnRH antagonist cetrorelix we conducted a 5 day treatment of peripubertal male rats (cetrorelix group n=12, 100 microg/d intraperitoneally injected; placebo n=10, NaCl 0.9% intraperitoneally injected) from postnatal day 32 to 36 and decapitated on postnatal day 37 to investigate the effects on pubertal development, serum gonadotropin and testosterone levels as well as the GnRH release from explanted hypothalami. A control group of 5 male rats was added for hypothalamus superfusion experiments on day 25. We observed no progress of testicular development in the cetrorelix group. Cetrorelix injected rats had lower testicular weights (531+/-13 versus controls 819+/-25 mg, mean+/-SEM, p<0.0001). 12 h after the last injection testosterone levels were in the castrate range (serum testosterone, median controls: 1.7 ng/ml, median cetrorelix <0.30 ng/ml, p<0.001), and they showed lower serum LH and FSH compared to the same age placebo group. After decapitation the preoptic mediobasal hypothalamic area (POA/MBH) was dissected from 5 randomly selected rats from each treatment group and the release rates of GnRH were determined in superfusion experiments: The hypothalamic GnRH secretion was comparable in the CET and the same age placebo rats but significantly higher than in the 25 day old control group. Conclusion: The GnRH antagonist cetrorelix inhibits the pituitary-gonadal axis in peripubertal male rats and may be effective in treating central precocious puberty in males.     

Effect of serotoninergic system on FSH secretion in male and female rats: evidence for stimulatory and inhibitory actions

The present experiments were designed to assess the effect of the serotoninergic system on FSH secretion in prepubertal (16-, 18-, 20-, 26- and 30-day-old) and adult (60-day-old) male and female rats. The intraperitoneal administration of 5-hydroxytryptophan (5-HTP), a serotonin (5-HT) precursor, induced a significant increase of FSH levels in male rats at 16, 26, 30 and 60 days of age (p less than 0.01) but not at 18 and 20 days. In the females, no modifications in FSH concentrations in prepubertal and adult rats were observed after 5-HTP injection. These results indicate that there are sexual differences in the effect of the serotoninergic system on FSH secretion. Orchidectomy performed in prepubertal (30-day-old) and adult rats (60 days) besides increasing FSH levels, reversed the response of this hormone to 5-HTP administration, since in castrated rats the 5-HT precursor induced a significant decrease in serum FSH concentrations. The administration of 5-HTP to 30 day-old castrated rats treated with testosterone induced FSH release as it did in control animals of this age. In the adult castrated rats the administration of testosterone abolished the inhibitory effect of 5-HTP observed in castrated rats but did not invoke the stimulatory action of 5-HTP observed in the controls. This difference between prepubertal and adult rats appears to be related to a decrease in the sensitivity to testosterone of the central mechanism which controls FSH secretion. These results indicate that 5-HTP stimulates FSH release in normal rats and inhibits FSH release in castrated rats, testosterone concentration being a relevant factor in determining the 'type' of effect produced. The hypothalamic concentrations of 5-HT and 5-hydroxy-indoleacetic acid (5-HIAA) are significantly higher at the ages at which 5-HTP induces FSH release than at other ages suggesting that hypothalamic turnover of these substances is related is related to the releasing effect of 5-HTP on FSH.     

Elevating circulating leptin in prepubertal male rhesus monkeys (Macaca mulatta) does not elicit precocious gonadotropin-releasing hormone release,assessed indirectly

The purpose of this study was to examine the hypothesis that the pubertal reaugmentation of pulsatile GnRH release in male primates is triggered by a rise in circulating leptin concentrations. Agonadal juvenile male rhesus monkeys (n = 7) were implanted with indwelling venous catheters and housed in specialized cages that allow continuous access to the venous circulation. GnRH release was monitored indirectly using LH secretion from the in situ pituitary sensitized to the LH releasing action of GnRH as a bioassay for the hypothalamic peptide. Infusion of recombinant human leptin (5 micro g/kg body weight.h for 16 d resulted in a marked square wave increment in circulating leptin concentration from approximately 2-20 ng/ml but did not elicit precocious GnRH release. GH secretion, however, was stimulated confirming that the heterologous leptin preparation was bioactive in the monkey. Parenthetically, recombinant human leptin was found to be immunogenic in the monkey and circulating antileptin IgG was demonstrable 22-35 d after the initial exposure to the human protein. These findings further support the view that circulating leptin is unlikely to provide the signal that triggers the onset of puberty in male primates.     

Sustained intermittent release of gonadotropin-releasing hormone in the prepubertal male rhesus monkey induced by N-methyl-DL-aspartic acid

The purpose of the present study was to determine whether gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus of the prepubertal monkey may be prematurely provoked into producing a sustained train of intermittent GnRH release N-methyl-DL-aspartic acid (NMA), an analog of the putative excitatory neurotransmitter aspartate, was used to stimulate the hypothalamus. In order to utilize pituitary luteinizing hormone (LH) secretion as a bioassay of hypothalamic GnRH release, juvenile males were castrated and the responsiveness of their gonadotrophs to GnRH was enhanced prior to the study with a chronic intermittent intravenous infusion of the synthetic decapeptide (0.1 microgram/min for 3 min every hour). Treatment with this regimen of GnRH, which appears to provide the pituitary gonadotrophs with a hypophysiotropic stimulus similar to that produced by the hypothalamus of castrated adults, elicited a pattern of pulsatile LH secretion in prepubertal animals similar to that observed in the open-loop situation in adults. This episodic pattern of LH release was sustained without decrement following termination of GnRH priming and initiation of an intermittent intravenous infusion of NMA (4.5-6.5 mg NMA/kg body weight/pulse, administered over 1 min) delivered at a frequency of 1 pulse/1 h for 50 h. In contrast, an intermittent infusion of the vehicle employed to administer NMA (saline) failed to maintain LH secretion. Administration of the same dose of NMA at a slower frequency of 1 pulse/2 h for 52 h, while also sustaining LH secretion without decrement, resulted in an exaggeration in the LH response.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-methyl-D,L-aspartate elicits hypothalamic gonadotropin-releasing hormone release in prepubertal male rhesus monkeys (Macaca mulatta)

In higher primates, the protracted delay from infancy to puberty results from an interruption in hypothalamic GnRH release. To determine whether the quiescent hypothalamic GnRH neurons of the prepubertal macaque are capable of discharging the decapeptide in response to a generalized neural depolarization, an excitatory amino acid analog, N-methyl-D,L-aspartate (NMA), was administered systemically to orchidectomized rhesus monkeys between 13 and 20 months of age. GnRH secretion was estimated indirectly by monitoring changes in circulating LH concentrations after the responsivity of pituitary gonadotropes to GnRH had been greatly facilitated by the chronic intermittent iv infusion of GnRH (0.1 microgram/min for 3 min every hour). The iv bolus administration of increasing doses of NMA (1.5, 4.8, and 15.0 mg/kg BW), 10-14 h after termination of the priming infusion of GnRH, elicited distinct discharges of LH, with magnitudes directly related to the amount of the excitant injected. Administration of a higher dose of NMA (48 mg/kg BW), however, failed to induce further LH release. The finding that pretreatment with a long-acting and potent GnRH receptor antagonist [( AcD2Nal1,4ClPhe2,DTrp3,DArg6,DAla10] GnRH-HOAc) abolished the LH-releasing activity of NMA provides compelling evidence for the view that the action of the neural excitant to induce gonadotropin release was exerted at a suprapituitary level. The additional observation that an N-methyl-D-aspartate receptor antagonist (D,L-2-amino-5-phosphono-valeric acid) blocked the NMA-induced release of GnRH suggests that the amino acid analog interacted with the N-methyl-D-aspartate receptor on neurons that synthesize and/or control the release of the hypothalamic hormone. Most interestingly, three sequential GnRH discharges, with a period and an amplitude apparently similar to those generated by the hypothalamus of the adult, were elicited from the brain of prepubertal monkeys by the sequential administration of three injections of NMA at hourly intervals. Taken together these findings demonstrate that the apparent dormancy of hypothalamic GnRH neurons, which is characteristic of prepubertal development in higher primates and underlies the protracted delay in the onset of puberty in these species, may be readily terminated by application of a generalized neural excitation. Plasma FSH, PRL, GH, and cortisol concentrations were also monitored during the course of some of these experiments, and release of each of these four hormones was observed after the iv injection of NMA (15 mg/kg BW).