Puberty presents remarkable individual differences in timing reaching over 5 years in humans. We put emphasis on the two edges of the age distribution of pubertal signs in humans and point to an extended distribution towards earliness for initial pubertal stages and towards lateness for final pubertal stages. Such distortion of distribution is a recent phenomenon. This suggests changing environmental influences including the possible role of nutrition, stress and endocrine disruptors. Our ability to assess neuroendocrine effects and mechanisms is very limited in humans. Using the rodent as a model, we examine the impact of environmental factors on the individual variations in pubertal timing and the possible underlying mechanisms. The capacity of environmental factors to shape functioning of the neuroendocrine system is thought to be maximal during fetal and early postnatal life and possibly less important when approaching the time of onset of puberty.

In order to evaluate the involvement of gonadotropin-releasing hormone (GnRH) in the effects of neuroexcitatory amino acids on luteinizing hormone (LH) secretion, N-methyl-D,L-aspartate (NMDA; 30 mg/kg s.c.) was administered to 50-day-old male rats. The in vitro release of GnRH from the hypothalamus showed a maximal increase (4.6-fold) in animals sacrificed 7.5 min after NMDA injection, while serum LH levels rose concomitantly. Incubation of rat hypothalami in vitro with kainate or NMDA concentrations > 0.1 mM resulted in a dose-related release of GnRH, NMDA being twofold more potent than kainate. Quisqualate (10 mM) did not affect the release of GnRH. On a molar basis, quinolinate (50 mM), a possible endogenous ligand for NMDA receptors, was the most effective in inducing GhRH secretion (34.9 ± 4.9 pg/7.5 min, mean increment ± SEM, n= 10). The effects of kainate and NMDA were mediated through different types of receptors, since GnRH response to kainate was unchanged in the absence of glycine or in the presence of increased concentrations of Mg²⁺ (2 mM) or Ca²⁺ (5.8 mM). In contrast, the GnRH response to NMDA was reduced by Ca²⁺ (5.8 mM) and abolished in the absence of glycine or in the presence of Mg²⁺ (2 mM). In addition, DL-amino-5-phosphonopentanoic acid (AP5), a competitive antagonist of NMDA receptors, prevented the NMDA-induced release of GnRH. The permissive effect of glycine on GnRH response to NMDA was 2.7-fold more important using glycine concentrations of 0.01 µM than when concentrations >100 µM were used. Intermittent incubation with NMDA in vitro (every other 7.5-min period) did not affect the amplitude of GnRH response, while continuous exposure to NMDA resulted in an initial and transient increase in GnRH release followed by a prolonged desensitization period. It is concluded that different neuroexcitatory amino acids acting through distinct receptor types may be involved in the hypothalamic control of LH release by regulating the secretion of GnRH.

An increase in the frequency of pulsatile gonadotropin-releasing hormone (GnRH) secretion in vitro and a reduction in LH response to GnRH in vivo characterize hypothalamic-pituitary maturation before puberty in the female rat. In girls migrating for international adoption, sexual precocity is frequent and could implicate former exposure to the insecticide dichlorodiphenyltrichloroethane (DDT), since a long-lasting DDT derivative has been detected in the serum of such children. We aimed at studying the effects of early transient exposure to estradiol (E(2)) or DDT in vitro and in vivo in the infantile female rat. Using a static incubation system of hypothalamic explants from 15-day-old female rats, a concentration- and time-dependent reduction in GnRH interpulse interval (IPI) was seen during incubation with E(2) and DDT isomers. These effects were prevented by antagonists of alpha-amino-3-hydroxy-5-methylisoxazole-4 propionic acid (AMPA)/kainate receptors and estrogen receptor. Also, o,p'-DDT effects were prevented by an antagonist of the aryl hydrocarbon orphan dioxin receptor (AHR). After subcutaneous injections of E(2) or o,p'-DDT between Postnatal Days (PNDs) 6 and 10, a decreased GnRH IPI was observed on PND 15 as an ex vivo effect. After DDT administration, serum LH levels in response to GnRH were not different from controls on PND 15, whereas they tended to be lower on PND 22. Subsequently, early vaginal opening (VO) and first estrus were observed together with a premature age-related decrease in LH response to GnRH. After prolonged exposure to E(2) between PNDs 6 and 40, VO occurred at an earlier age, but first estrus was delayed. We conclude that a transient exposure to E(2) or o,p'-DDT in early postnatal life is followed by early maturation of pulsatile GnRH secretion and, subsequently, early developmental reduction of LH response to GnRH that are possible mechanisms of the subsequent sexual precocity. The early maturation of pulsatile GnRH secretion could involve effects mediated through estrogen receptor and/or AHR as well as AMPA/kainate subtype of glutamate receptors.

In the male rat the timing of puberty can be estimated by the rapid increase in testicular weight occurring between 25-50 days of age. We found that elongated spermatids, the most mature germ cells identified using flow cytometry, were first seen at 25 days (4% of the testicular cells), while an adult proportion (63%) was attained by 45 days of age. We have shown previously that hypothalamic explants could release GnRH in a pulsatile fashion at a frequency increasing around the age of 25 days, thus consistent with the time of onset of puberty. Since pulsatile GnRH secretion could be suppressed by MK-801, a noncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptor activation, we postulated that an increased activation of those receptors could be involved in the neuroendocrine mechanism that activates pulsatile GnRH secretion at the onset of puberty. Such a concept was supported by the NMDA-induced release of GnRH, which was observed using 1 mM NMDA at 25 days, while a dose of 20-50 mM was required at 15 or 50 days of age. MK-801 could provide an index of NMDA receptor activation, since the antagonistic effect of MK-801 is use dependent. This particular property was confirmed by the inability of MK-801 (5 pM) to block the depolarization (veratridine)-induced release of GnRH in the presence of 0.001 mM NMDA, while partial or complete suppression was obtained in the presence of 0.1 and 10 mM NMDA, respectively. Using explants obtained at 5, 10, 15, 20, 25, 30, 35, and 50 days of age, the lowest concentrations of MK-801 that blocked the veratridine-induced release of GnRH were, respectively, 10(7), 10(7), 10(7), 10(3), 10, 10(2), 10(4), and 10(8) pM. In contrast, there was no age-related difference in sensitivity to the inhibitory effect of Mg2+, a noncompetitive NMDA receptor antagonist which is not use dependent. The pulsatile secretion of GnRH occurred at a similar frequency at 25 and 50 days of age (4.7 and 5.4 pulses/3.5 h, respectively) but it was suppressed by a lower MK-801 concentration at 25 days (10(4) pM) than at 50 days (10(8) pM). These data indicate that the NMDA receptors involved in the control of pulsatile GnRH secretion are markedly and transiently activated around the time of onset of puberty in the male rat.

The present study aimed to investigate the effects of leptin and ghrelin on pulsatile pulsatile gonadotrophinreleasing hormone (GnRH) secretion in vitro with emphasis on neuropeptide mediators and changes between prepuberty (15 days) and sexual maturity (50 days) in the male rat. When hypothalamic expiants were studied 90 min after an intraperitoneal injection of leptin, ghrelin or agouti-related protein (AgRP) at 15 days, the GnRH interpulse interval (IPI) was significantly increased by ghrelin and AgRP and decreased by leptin. At 50 days, an increase in GnRH IPI was also caused by ghrelin and AgRP. When the peptides were directly incubated with the expiants, the effects of leptin and AgRP in vitro were consistent with those seen after in vivo administration. By contrast, ghrelin resulted in a reduction of GnRH IPI and this was observed at 15 days only. To delineate the neuropeptide mediators of leptin and the effects of ghrelin in the hypothalamus, various hypothalamic neuropeptides and antagonists were used in vitro. At 15 days, the GnRH IPI was significantly decreased after incubation with cocaine and amphetamine-regulated transcript (CART), α-melanocyte-stimulating hormone, corticotrophin-releasing factor (CRF) and neuropeptide Y (NPY). The reduction of GnRH IPI caused by leptin was partially prevented by either an anti-CART antiserum or SHU 9119, a melanocortin MC3/MC4 receptor antagonist or a CRF receptor antagonist. The NPY-Y5 receptor antagonist did not influence the effects of leptin whereas that antagonist totally prevented the decrease in GnRH IPI caused by ghrelin. The ghrelin-induced reduction of GnRH IPI was partially prevented by SHU 9119. When used alone, SHU 9119 or a CRF-receptor antagonist resulted in increased GnRH IPI at 50 days while they had no effects at 15 days. The NPY-Y5 receptor antagonist resulted in increased GnRH IPI at 15 and 50 days. In conclusion, leptin and ghrelin show opposing effects on pulsatile GnRH secretion after administration in vivo whereas they both have stimulatory effects in vitro. Such effects involve consistently the anorectic peptides CART and CRF for leptin that are mainly active at 15 days. The melanocortigenic system appears to mediate the effects of both leptin and ghrelin. The effects of ghrelin also involve NPY receptors and operate effectively before and at sexual maturity.Key words: gonadotrophin-releasing hormone, leptin, ghrelin, AgRP, α-MSH, CRF, rat.In the hypothalamus, the control of energy balance is tightly regulated through complex interactions between peripheral nutrition-related hormones and hypothalamic neuropeptides (1, 2). Several decades ago, a link between fat mass and menstrual cyclicity already suggested that nutrition-related hormones could influence the hypothalamic control of puberty and reproduction (3). Further insight came from isolation of leptin, an adipocyte-derived anorectic hormone involved in the regulation of body weight, as well as a facilitatory or permissive control of puberty and reproduction (4, 5). We have pre...

Our aim was to study the effect of estradiol (E2) on pulsatile GnRH secretion in vitro in relation to sex and development. When hypothalamic explants obtained from 5- and 15-d-old female rats were exposed to E2 (10(-7) m), a reduction of GnRH interpulse interval (IPI) occurred but not at 25 and 50 d of age. This effect was prevented by the estrogen receptor antagonist ICI 182.780 and the AMPA/kainate receptor antagonist DNQX but not by the AMPA and N-methyl-d-aspartate receptor antagonists SYM 2206 and MK-801. E2 did not affect GnRH IPI in hypothalamic explants obtained from male rats. Therefore, the possible relation between the female-specific effects of E2 in vitro and perinatal sexual differentiation was investigated. When using explants obtained from female rats masculinized through testosterone injection on postnatal d 1, E2 was no longer effective in vitro at 5 and 15 d. In addition, with explants obtained from male rats demasculinized through perinatal aromatase inhibitor treatment, E2 became capable of decreasing GnRH IPI in vitro at 15 d. To study the possible pathophysiological significance of early hypothalamic E2 effects, female rats received a single E2 injection on postnatal d 10. This resulted in reduced GnRH IPI in vitro on d 15 as well as advancement in age at vaginal opening and first estrus. In conclusion, E2 decreases the GnRH IPI in the immature female hypothalamus in vitro through a mechanism that depends on perinatal brain sexual differentiation and that could be involved in some forms of female precocious puberty.

Leptin may act as a negative feedback signal to the hypothalamic control of appetite through suppression of neuropeptide Y (NPY) secretion and stimulation of cocaine and amphetamine regulated transcript (CART). We aimed at studying the effects of leptin, CART and NPY on the hypothalamic control of the pituitary-gonadal system. Pulsatile gonadotropin-releasing hormone (GnRH) secretion was studied in vitro using retrochiasmatic hypothalamic explants from adult rats. In the female, GnRH pulse amplitude was significantly 27 26increased by leptin (10 M) and CART (10 M) irrespective of the estrus cycle phase while no such effects were seen in the male. The GnRH interpulse interval was not affected in both sexes. Passive immunoneutralization against CART caused a reduction in GnRH pulse 27 amplitude in the female. A slight but significant increase in GnRH pulse amplitude was caused by NPY (10 M) in the female. However, 26GnRH pulse amplitude was not affected by a Y5-receptor antagonist (10 M) while the interpulse interval was significantly increased as shown previously in the male. The increase in GnRH pulse amplitude caused by leptin was totally prevented by coincubation with an 27 anti-CART antiserum whereas it was not affected by coincubation with the NPY Y5-receptor antagonist (10 M). In conclusion, leptin and NPY show separate permissive effects on GnRH secretion in the adult rat hypothalamus. In both sexes, NPY is prominently involved in the control of the frequency of pulsatile GnRH secretion through the Y5 receptor subtype. Leptin causes a female-specific facilitatory effect on GnRH pulse amplitude which is mediated by CART and which occurs irrespective of the estrus cycle phase.

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.