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Endocrinology Vol. 141, No. 4 1464-1469
Copyright © 2000 by The Endocrine Society

ARTICLES

In Vitro Stimulation of the Prepubertal Rat Gonadotropin-Releasing Hormone Pulse Generator by Leptin and Neuropeptide Y through Distinct Mechanisms1

M. C. Lebrethon, E. Vandersmissen, A. Gérard, A. S. Parent, J. L. Junien and J. P. Bourguignon

Developmental Neuroendocrinology Unit (M.C.L, E.V, A.G, A.S.P, J.P.B.), Division of Ambulatory Pediatrics and Adolescent Medicine, Department of Pediatrics, University of Liège, C.H.U. Sart Tilman, B-4000 Liège, Belgium; and Ferring Research Institute (J.L.J), Paris 75007, France

Address all correspondence and requests for reprints to: Marie-Christine Lebrethon, M.D., Ph.D., Division of Pediatric and Adolescent Medicine, C.H.U. Sart Tilman, B35, B-4000 Liège, Belgium. E-mail: Marie-Christine.Lebrethon{at}chu.ulg.ac.be


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Leptin may act as a negative feedback signal to the brain in the control of appetite through suppression of neuropeptide Y (NPY) secretion and stimulation of cocaine- and amphetamine-regulated transcript (CART), a new anorectic peptide. We aimed at studying whether leptin, NPY, and CART have related effects on the hypothalamic control of the pituitary-gonadal system and the developmental changes in NPY and CART effects. Using retrochiasmatic hypothalamic explants from prepubertal 15-day-old male rats, the GnRH interpulse interval (mean ± SD : 62 ± 5 min) was significantly reduced by 10-7 M of leptin (46 ± 3.3 min) as well as 10-7 M of NPY (47 ± 4.4 min) and 10-6 M of CART (46 ± 2.7 min), whereas the GnRH pulse amplitude was not affected. The stimulatory effects of different NPY receptor agonists [human PYY 3–36, porcine NPY 13–36, human (D-Trp 32) NPY, porcine (Leu 31 Pro 34) NPY, human pancreatic polypeptide (PP)], as well as the absent effects of rat PP were consistent with the involvement of the Y5-receptor subtype in mediation of NPY effects. Incubation with 10-7 M of a Y5-receptor selective antagonist prevented the effect of NPY (61 ± 4 vs. 46 ± 2 min), whereas leptin and CART effects were not (47 ± 3 vs. 46 ± 3 min and 46 ± 3 vs. 46 ± 2 min, respectively), suggesting that NPY was not involved in leptin and CART effects. Using an anti-CART antiserum (1:1000), the reduction of GnRH interpulse interval caused by leptin was partially prevented (56.2 ± 4 vs. 47.9 ± 3.8 min), whereas the reduction of GnRH interval caused by NPY was not affected (45.9 ± 2.5 vs. 47.8 ± 3.7). The GnRH interpulse interval was decreased by 10-7 M of NPY at 5 days (72 ± 3.8 vs. 91.9 ± 3.5) as well as at 15 days, whereas such an effect was not observed anymore at 25 and 50 days. Similar effects were observed using 10-6 M of CART-peptide. Using 10-6 M of the Y5-receptor antagonist, the GnRH interpulse interval was significantly increased at 15 days (66.6 ± 2.7 min), 25 days (56.5 ± 39.9 min), and 50 days (52.5 vs. 38.2 min), whereas no change was observed at 5 days. Using the anti-CART antiserum, a significant increase of GnRH interpulse interval was observed at 25 days only. In conclusion, the stimulatory effects of leptin and NPY on the frequency of pulsatile GnRH secretion before puberty involve two distinct mechanisms. NPY causes acceleration of GnRH pulsatility via the Y5-receptor subtype, which is not involved in leptin effects while the CART is involved in leptin effects on GnRH secretion but not in NPY effects. The reduction of pulsatility by the Y5 antagonist provides evidence of endogenous NPY involvement in the control of GnRH secretion from the time of onset of puberty.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LEPTIN, WHICH IS produced by adipose cells, circulates in concentrations proportional to adiposity and serves as a negative feedback signal to the hypothalamus through suppression of appetite and stimulation of energy expenditure (1). In addition, leptin may act as a metabolic signal involved in the control of puberty and reproduction. Chronic leptin treatment can restore fertility in the leptin deficient ob/ob mice of both sexes (2, 3). In the normal mouse (4, 5) and rat (6), leptin has been shown to advance the onset of puberty and to increase GnRH secretion in vitro (7, 8). Neuropeptide Y (NPY), a potent stimulator of food intake, has been proposed to mediate the hypothalamic effect of leptin. In food-restricted rats, the NPY messenger RNA (mRNA) levels in the hypothalamus are increased (9). In fasted rats, intracerebroventricular injections of leptin reduce hypothalamic NPY mRNA levels as well as NPY levels in the arcuate nucleus (10, 11). In the leptin deficient ob/ob mice, the overexpression of NPY in the arcuate nucleus (12, 13) is attenuated by leptin administration (13, 14). The leptin receptor and preproNPY mRNA are coexpressed in the mouse arcuate nucleus (15).The role of NPY in the hypothalamic control of the pituitary-gonadal axis is more complex and controversial. Discrepant stimulatory or inhibitory effects on sexual maturation and reproduction have been observed depending on species, steroidal environment (16), site of NPY administration into the brain (17) and chronic (18, 19, 20) vs. acute pattern of infusion (21).

Here, we aimed at studying leptin and NPY effects on pulsatile GnRH secretion from rat hypothalamic explants. Because we found that both leptin and NPY were involved in the acceleration of pulsatile GnRH secretion preceding the onset of puberty in male rat, we attempted to determine whether leptin and NPY were involved in a common neural pathway. Therefore, we characterized the NPY-Y subtype of receptor mediating NPY effects and we studied there possible involvement in leptin effects. The cocaine and amphetamine-regulated transcript (CART)-peptide (22) was shown recently to be an endogenous inhibitor of food intake in mice and rat, this effect being regulated by leptin (23). We showed recently that CART mediated the acceleration of pulsatile GnRH secretion mediated by leptin (24). Here, we examined the possible role of CART in the effects of NPY on GnRH pulsatility.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male Wistar rat were used. They were housed in temperature- and light-controlled conditions with water and standard rat pellets ad libitum. The prepubertal pups were with lactating mothers until weaning which was at 3 weeks of age. The protocols were approved by the University Committee on Animal Research.

Hypothalamic explant incubation and GnRH RIA
The animals were killed by decapitation between 1000 and 1100 h. The retrochiasmatic hypothalamus was rapidly dissected and transferred into a static incubator as described previously (25, 26). In each experiment, 12 to 15 explants were studied individually for 4 to 6 h through collection and renewal of the incubation medium (0.5 ml) every 7.5 min. The samples were frozen until assayed. GnRH was measured using a highly sensitive RIA (25, 26). The values below the limit of detection (5 pg/7.5 min) were assigned that value. Two different GnRH antisera were used, generously gifted by Dr. A. W. Root (St. Petersburg, FL) (27) and by Dr. Y. F. Chen and V. D. Ramirez (Urbana, IL) (28). Both antisera were highly specific for GnRH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) without significant cross-reactivity of neither GnRH (1, 2, 3, 4, 5, 6, 7, 8, 9) and GnRH (1, 2, 3, 4, 5), nor any of the reagents used in the experiments.

Study protocols
Using retrochiasmatic hypothalamic explants obtained at day 15, the frequency and the amplitude of pulsatile GnRH secretion was studied without (control) or with different treatment. Mouse recombinant leptin (R&D Systems, Abingdon, UK) and porcine NPY (Ferring Pharmaceuticals Ltd., Copenhagen, Denmark) were used at concentrations between 10-10 and 10-6 M. The CART (52–102) -peptide (Novo Nordisk, Bagsvaerd, Denmark) was used at 10-6 M and an anti-CART antiserum (Novo Nordisk) was used at a 1 :1000 dilution.

To determine the NPY Y receptor subtype involved in NPY effects, six NPY agonists were used. Porcine NPY 13–36 10-7 M, porcine (Leu 31 Pro 34) NPY 10-7 M, human (D-Trp32) NPY 10-7 M and 10- 6 M, human pancreatic polypeptide (PP) 10-7 M, human polypeptide YY (PYY) (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) 10-7 M and 3 10-7 M, were purchased from Neosystem (Strasbourg, France) and rat PP, 10-7 M was from Sigma (St. Louis, MO). Further studies on NPY-Y1-and Y5-receptors were performed through incubation using selective Y1 antagonist (BIBO 3304 TF, Boehringer Ingelheim Pharma KG, Biberach, Germany) and Y5 antagonist, trans-naphthalene-1-sulfonic acid-4-{(4-(3-dimethylamino-propylamino)-quinazolin-2-ylamino)-methyl}-cyclohexylmethyl}-amide (29), which was synthesized in the Medicinal Chemistry Department of Ferring Pharmaceuticals Ltd. Research Ltd. (Chilworth, UK). The explants were incubated with NPY 10-7 M alone or combined with the Y1 or the Y5 receptor antagonist (10-7 M).

To determine the signaling pathway of NPY and leptin, we studied the effects of an anti-CART antiserum (1:1000) or the NPY-Y5 antagonist (10-7 M) on the NPY (10-7 M)-, leptin (10-7 M)- and CART (10-6 M)-induced changes in pulsatile GnRH secretion.

To study whether an endogenous NPY tone is developmentally involved in the hypothalamic control of pulsatile GnRH secretion, hypothalamic explants from 5-, 15-, 25-, and 50-day-old male rat were incubated with NPY (10-7 M) or with the NPY Y5 receptor antagonist (10-6 M). In similar age and experimental conditions, hypothalamic explants were incubated with CART (10-6 M) or with an anti-CART antiserum (1:1000).

Statistical analysis
The occurrence of significant pulses of GnRH secretion was determined using the Pulsar program as described previously (30). The individual interpulse interval and pulse amplitude as well as the mean ± SD were calculated. The effects of increasing doses of leptin and NPY on pulsatile GnRH frequency were analyzed by one-way ANOVA followed by linear regression test. For the other studies, one-way ANOVA was followed by Student’s-Newman-Keuls test. The threshold for statistical significance was at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of leptin and NPY on frequency and amplitude of pulsatile GnRH secretion
Using explants from 15-day-old male rats, the GnRH interpulse interval was significantly decreased by leptin and NPY in a similar dose-related manner (Fig. 1Go) whereas the GnRH pulse amplitude was not affected (12.3 ± 7.3 vs. 12.8 ± 5.9 pg/7.5 min, leptin 10-7 M vs. control and 15.7 ± 7.7 vs. 15.7 ± 7.9 pg/7.5 min, NPY 10-7 M vs. control).



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  		Figure 1. Effects of increasing concentration of leptin and NPY on the frequency of pulsatile GnRH secretion using hypothalamic explants from 15-day-old male rats. * P < 0.05 vs. controls.

 
Characterization of NPY Y subtype receptor mediating NPY effects and interaction with leptin effects
A significant stimulatory effect on the frequency of pulsatile GnRH secretion from hypothalamic explant of 15-day-old male rat was observed using 10-7 M of different NPY analogs : porcine NPY 13–36, porcine (Leu 31, Pro 34) NPY, human (D-Trp 32) NPY, human PYY 3–36, and human PP (Table 1Go). In all these conditions, the effect was significantly less than that obtained using 10-7 M of porcine NPY. Higher concentrations of human PYY 3–36 or human (D-Trp32) NPY resulted in a greater reduction of the interpulse interval, which was not different anymore from the effects of 10-7 M of NPY. No stimulatory effect on pulsatile GnRH secretion was observed using 10-7 M of rat PP. This profile of agonist activity was found to be consistent with the involvement of the Y5-receptors. Incubation of 15-day-old explants with a Y5-receptor antagonist (10-7 M) was able to prevent the stimulatory effect of NPY (10-7 M) on the frequency of pulsatile GnRH secretion, whereas the antagonist did not show any effect when used alone (Table 2Go). Using the Y1-receptor antagonist, the effects of 10-7 M of NPY could not be prevented.


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  		Table 1. Characterization of NPY Y receptor subtype mediating NPY effects on frequency of pulsatile GnRH secretion through incubation of hypothalamic explants from 15-day-old male rats with different NPY receptors agonists

 

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  		Table 2. Effects of NPY Y1- and Y5-receptor antagonist on the reduction of the GnRH interpulse interval caused by NPY

 
Using hypothalamic explants from 15-day-old male rats, the GnRH interpulse interval was significantly decreased by 10-6 M of CART-peptide, and the effect was similar to that of 10-7 M of NPY and leptin (Fig 2Go). Using an anti-CART antiserum (1:1000), the effect of CART on GnRH pulsatility can be totally prevented. The anti-CART antiserum can significantly but not totally overcome leptin effects on GnRH interpulse interval, whereas the reduction of GnRH interpulse interval caused by 10-7 M of NPY was not affected (Fig 2Go). In contrast, incubation with 10-7 M of the Y5-receptor antagonist prevented the effect of NPY 10-7 M on the GnRH interpulse interval whereas the effects of leptin 10-7 M or CART 10-6 M were not affected (Fig 2Go).



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  		Figure 2. Effects of anti-CART antiserum (AS) or NPY Y5-receptor antagonist (Y5-ant) on the increase in frequency of pulsatile GnRH secretion induced by CART, leptin, or NPY using hypothalamic explants from 15 day-old male rats. *, P < 0.05 vs. control. &, P < 0.05 vs. peptide.

 
Developmental changes of NPY and CART effects
At 5 days and 15 days, the GnRH interpulse interval was decreased by 10-7 M of NPY (Fig. 3AGo) and 10-6 M of CART (Fig. 3BGo), whereas such an effect was no longer observed at 25 and 50 days. Using 10-6 M of the Y5-receptor antagonist, the GnRH interpulse interval was significantly increased at 15, 25, and 50 days, whereas no change was observed at 5 days (Fig. 3AGo). Using 1:1000 dilution of anti-CART antiserum, a significant increase of GnRH interpulse interval was observed at 25 days only (Fig. 3BGo).



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  		Figure 3. Effects of NPY and NPY-Y5 receptor antagonist (upper panel) and CART and anti-CART antiserum (lower panel) on the frequency of pulsatile GnRH secretion from hypothalamic explants (n = 4–12) of male rats at different ages. * P < 0.05 vs. control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In many species, including man, acquisition and maintenance of reproductive capacity is dependent on the activity of the hypothalamic GnRH pulse generator (31). Because the initiation of puberty as well as the integrity of reproductive function were physiologically coupled to nutritional status, it was logical to postulate that leptin and NPY contributed to the hypothalamic control of pulsatile GnRH secretion. Based on recent literature data, such a hypothesis appeared unequivocally to be true for a stimulatory role of leptin. In the female rat, ip (7) or intracerebroventricular (8) administration of leptin was shown to increase LH levels, whereas leptin antiserum administration caused decrease in plasma LH and disrupted estrous cyclicity (32). In normal female mice, early onset of reproductive function was observed following leptin treatment (4, 5). In an other study, using female hypothalamic explants, we have shown that leptin caused increase in frequency of pulsatile GnRH secretion before puberty and increase in amplitude after puberty (24). In this study, using hypothalamic explants from prepubertal male rats, we show a dose-related stimulatory effect of leptin on the frequency of pulsatile GnRH secretion. These data confirm that leptin is involved in the facilitatory regulation of GnRH secretion. As far as NPY, evidence accumulated that this peptide could affect GnRH/LH secretion, but the effects were either stimulatory or inhibitory depending on several factors. Steroid environment was shown to be important in rodents and rabbits with an increase of GnRH secretion in intact animals and a decrease in the absence of gonadal steroids (16, 33, 34). The site of NPY administration and the acute vs. chronic exposure may also account for different effects. In the monkeys, NPY was stimulatory when acutely injected in the median eminence area (35), whereas NPY was inhibitory when acutely or chronically administered in the third ventricle (17, 36). In this paper, we show that acute in vitro exposure of prepubertal rat hypothalamic explants to NPY increases pulsatile GnRH secretion in a dose-related manner suggesting a permissive effect of NPY on GnRH secretion before puberty. In rats, chronic intracerebroventricular administration of NPY could inhibit secretion in both sexes (19, 20), resulting in delayed maturation (19). NPY also inhibited the rapid catch-up growth and sexual maturation occurring after discontinuation of food restriction in rats (18).

The actions of NPY are mediated through distinct receptor subtypes that show different affinity for NPY agonists and have been cloned (37). Both the Y5-receptor (38, 39) and the Y1-receptor subtype (40) were shown to be involved in NPY-induced food intake. Few studies were available regarding NPY receptor subtype involved in the hypothalamic control of the pituitary-gonadal axis. The activation of NPY receptors of the Y1 subtype was shown to be required for the physiological amplification of the spontaneous preovulatory LH surge in rats (41) as well as the increase in GnRH mRNA caused by NPY in the preoptic area (42). In our study, the results obtained with different NPY agonists were consistent with the involvement of the Y5 receptor subtype in the stimulatory effect on pulsatile GnRH secretion. The effect of these NPY agonists was characterized previously using cloned Y-type receptors based on negative coupling to cAMP concentrations (43). The Y2 receptor involvement could be ruled out by the stimulatory effect of porcine (Leu 31 Pro34) NPY. The Y4 receptor was not involved based on the stimulatory effect of human PP and the ineffectiveness of rat PP. It was unlikely that the Y1 receptor was involved because GnRH secretion was stimulated by both human PP and human PYY 3–36 which were weak or inactive Y1-receptor agonist in vitro. In addition, GnRH secretion induced by porcine NPY was not reduced by incubation with the Y1-receptor selective antagonist, BIBO 3304 TF. The role of the Y5- receptor in mediating NPY effects on pulsatile GnRH secretion was supported by the stimulatory effects of human PYY 3–36, porcine NPY and human PP which are potent Y5-receptor agonists in vitro. Human (D-Trp 32) NPY, a weak but selective Y5 agonist could elicit GnRH secretion as well. Ultimately, the Y5-receptor selective antagonist was able to prevent the NPY effects on pulsatile GnRH secretion, thus confirming the role of that particular receptor subtype. The NPY-Y5 receptor subtype was also shown to be involved in the inhibitory effects of NPY on the gonadotropic axis that were observed in vivo (44).

It has been proposed that NPY mediates some of the effects of leptin in the control of feeding behavior as well reproductive function. Leptin receptor were shown to be expressed by NPY neurons in the arcuate nucleus (15, 45), and NPY gene transcription was attenuated by leptin administration in ob/ob mice (46). In adverse metabolic conditions, reproductive function was impaired or reduced, and increased NPY gene expression was associated with low levels of leptin (47), suggesting that NPY was the link between leptin and GnRH secretion. Here, we show that both leptin and NPY have similar stimulatory effects on GnRH secretion that are in contrast to the opposite effects in the control of feeding behavior. More importantly, we show that different pathways are involved in the effects on GnRH secretion. Blockade of the NPY Y5 receptors that mediate NPY effects have no effect on leptin action indicating that NPY neurons are unlikely to mediate leptin effects. The involvement of other mediators in leptin action was suggested earlier by the observation that leptin was still effective in NPY knockout mice (48).

Recently, CART was shown to be an anorectic signal regulated by leptin because its expression in the hypothalamus was low in ob/ob mice and increased by leptin treatment (23). In another study, we showed recently that CART was a mediator of leptin effect on pulsatile GnRH secretion in the female rat (24). Here, we confirm those data in the prepubertal male rat. CART was also shown to markedly inhibit the NPY-induced feeding in fasted and normal rats, and immunocytochemical studies showed a close apposition of NPY-containing terminals with CART peptide-immunoreactive neurons in the hypothalamus (23), suggesting a possible NPY-CART communication line (40). Here, we have tested this hypothesis in the neuroendocrine control of pulsatile GnRH secretion in vitro. Using explants from male rat, we show that an anti-CART antiserum, which can partially prevent leptin effect, does not change the NPY effect on pulsatile GnRH secretion. Still, this suggests that different signaling pathways were used by leptin and NPY to accelerate GnRH pulsatility.

A role for NPY in sexual maturation was hypothesized because the hypothalamic NPY content steadily increased from birth to 31 days of age in the male rat (49). In the female rhesus monkey, immunoneutralization of endogenous NPY was shown to suppress pulsatile GnRH release during the midpubertal period, but not during the prepubertal period, suggesting a developmental change in the sensitivity of the GnRH network to NPY (50). In the male rat, we show here that NPY can increase pulsatile GnRH secretion at days 5 and 15, while such an effect is no longer observed at day 25 and 50. The effects obtained at 5 and 15 days may result from presence of receptors but absence of sufficient endogenous NPY tone, whereas the absent NPY effects at older ages may result from the already highly increased endogenous NPY tone at that time. Such a concept is consistent with the absent effects of the Y5-receptor selective antagonist at day 5, whereas a clear increase in pulsatile GnRH release is caused by the antagonist at day 15, 25, and 50. These in vitro data obtained in the male rat are somewhat discrepant from the data obtained in the female rhesus monkey in vivo, where NPY showed effects only after the onset of puberty (50). In the male rhesus monkey, NPY gene expression was shown to decrease during the peripubertal transition (51). Thus, though species differences may account for differences in the effect observed, there was an agreement on the existence of dramatic change in NPY effects at the time of sexual maturation. No data were available concerning the ontogeny of CART-peptide effects on pulsatile GnRH frequency. In the present study, we show that CART can increase pulsatile GnRH secretion at days 5 and 15, whereas no effect is observed at 25 and 50 days, suggesting a possible involvement of CART at the time of the onset of puberty. This hypothesis is consistent with the slight but significant increase of GnRH interpulse interval caused by the anti-CART antiserum at day 25. The absent anti-CART antiserum effects at day 50 may be explained either by a subtotal neutralization due to insufficient concentration of the antiserum. In addition, other mechanisms than CART mediation may be involved in leptin effects on pulsatile GnRH frequency.

In conclusion, we show that leptin and NPY are involved through distinct mechanisms in the acceleration of pulsatile GnRH secretion preceding the onset of puberty in the male rat. NPY causes acceleration of GnRH pulsatility via the Y5-receptor subtype that is not involved in leptin effects. CART, a new signaling peptide, is involved in the mechanism of leptin effects but not in NPY effects. In addition, the striking stimulatory effect of NPY in the prepubertal hypothalamus and inhibitory effect of the NPY-Y5-receptor subtype in the pubertal hypothalamus support the existence of a facilitatory NPY pathway activated at onset of puberty in the rat. Further studies are warranted to determine the targets neurons of leptin, NPY and CART involved in the hypothalamic mechanism of the onset of puberty.


   Acknowledgments
 
We would like to thank Dr. Peter Kristensen, Novo Nordisk A/S (Bagsvaerd, Denmark) for the generous gift of purified CART protein and CART antibody. We are grateful to Dr. Graeme Semple, Ferring Research Institute Ltd. (Chilworth, UK) for synthesis of the Novartis nonpeptidic Y5-receptor antagonist, and Dr. Wolfhand Engel, Boehringer Ingelheim Pharma KG (Biberach, Germany) for the generous supply of the Y1 antagonist BIBO 3304TF.


   Footnotes
 
1 This work was supported by grants from the Belgian Study Group for Pediatric Endocrinology, the Belgian "Fonds de la Recherche Scientifique Médicale" (grant 3.4529.97), the faculty of Medicine at the University of Liège, and Ferring Pharmaceuticals Ltd. Research Institute. Back

Received August 3, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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