[Endocrine disruption: a challenge in research, public health and clinical practice]. / La perturbation endocrinienne: entre enjeux de recherche, enjeux de santé publique et enjeux de pratique quotidienne.

Epidemiological and experimental data highlight the fetal and early postnatal life as critical periods for the effects of endocrine disrupting chemicals (EDCs), since exposure to EDCs during these periods can predispose to disease later in life. EDCs' effects include disorders of the reproductive system throughout life (abnormalities of sexual differentiation, infertility or subfertility and some neoplasia) and disorders of energy balance (obesity and metabolic syndrome). They could also influence the development of the cerebral cortex. However, the demonstration of the involvement of a single EDC remains difficult in human since we are virtually exposed to a mixture of several ubiquitous EDCs which are variably persistent in the environment and the body and have lifelong consequences. Moreover, since their dose-response relationship can be non-monotonic, setting a threshold dose for EDCs effects has become meaningless. Pregnant women, newborns and young children appear to be mostly at risk. However, the role of the physician remains difficult and raises several questions: how can we formulate justified, applicable and updated recommendations that are not counterproductive or alarmist...in a society that has to take the necessary steps to regulate production and protect the population?

Early neuroendocrine disruption in hypothalamus and hippocampus: developmental effects including female sexual maturation and implications for endocrine disrupting chemical screening.

The timing of puberty has been mainly studied in females for several reasons, including the possible evaluation of a precise timer (i.e. menarcheal age) and concerns with respect to the high prevalence of precocity in females as opposed to males. Human evidence of altered female pubertal timing after exposure to endocrine disrupting chemicals (EDCs) is equivocal. Among the limiting factors, most studies evaluate exposure to single EDCs at the time of puberty and hardly assess the impact of lifelong exposure to mixtures of EDCs. Some rodent and ovine studies indicate a possible role of foetal and neonatal exposure to EDCs, in accordance with the concept of an early origin of health and disease. Such effects possibly involve neuroendocrine mechanisms because the hypothalamus is a site where homeostasis of reproduction, as well as control of energy balance, is programmed and regulated. In our previous studies, pulsatile gonadotrophin-releasing hormone (GnRH) secretion control via oestrogen, glutamate and aryl hydrocarbon receptors was shown to be involved in the mechanism of sexual precocity after early postnatal exposure to the insecticide dichlorodiphenyltrichloroethane. Very recently, we have shown that neonatal exposure to the potent synthetic oestrogen diethylstilbestrol (DES) is followed by early or delayed puberty depending on the dose, with consistent changes in developmental increase of GnRH pulse frequency. Moreover, DES results in reduced leptin stimulation of GnRH secretion in vitro, an effect that is additive with prenatal food restriction. Thus, using puberty as an endpoint of the effects of EDC, it appears necessary to consider pre- and perinatal exposure to low doses and to pay attention to the other conditions of prenatal life, such as energy availability, keeping in mind the possibility that puberty could not only be advanced, but also delayed through neuroendocrine mechanisms.

Effects of in vivo and in vitro administration of ghrelin, leptin and neuropeptide mediators on pulsatile gonadotrophin-releasing hormone secretion from male rat hypothalamus before and after puberty.

The present study aimed to investigate the effects of leptin and ghrelin on pulsatile pulsatile gonadotrophin-releasing 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 explants 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 explants, 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), alpha-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.

Female sexual maturation and reproduction after prepubertal exposure to estrogens and endocrine disrupting chemicals: a review of rodent and human data.

Natural hormones and some synthetic chemicals spread into our surrounding environment share the capacity to interact with hormone action and metabolism. Exposure to such compounds can cause a variety of developmental and reproductive detrimental abnormalities in wildlife species and, potentially, in human. Many experimental and epidemiological data have reported that exposure of the developing fetus or neonate to environmentally relevant concentrations of some among these endocrine disrupters induces morphological, biochemical and/or physiological disorders in brain and reproductive organs, by interfering with the hormone actions. The impact of such exposures on the hypothalamic-pituitary-gonadal axis and subsequent sexual maturation is the subject of the present review. We will highlight epidemiological human studies and the effects of early exposure during gestational, perinatal or postnatal life in female rodents.

[Diabetes mellitus in childhood and adolescence. 1. Specific management by a multidisciplinary team]. / Le diabète de l'enfant et de l'adolescent. 1. Prise en charge par le Service Universitaire de Pêdiatrie et la Cellule "Education-Diabète en Pédiatrie".

The management and follow up of diabetes in youth is a multidisciplinary challenge due to both short and long term objectives. Awareness of the feelings and problems faced by the families is critical. The experience of our team has started in the 1960s and is briefly described and updated in this article.

Kainate/estrogen receptor involvement in rapid estradiol effects in vitro and intracellular signaling pathways.

Although the interactions between sex steroids and GnRH have been extensively studied, little is known about the mechanism of estradiol (E2) effects on GnRH secretion. In the present study, we used retrochiasmatic hypothalamic explants of 50-d-old male rats, and we observed that E2 significantly increased the glutamate-evoked GnRH secretion in vitro within 15 min in a dose-dependent manner. E2 also significantly increased the L-arginine-evoked GnRH secretion. E2 effects were time dependent because the initially ineffective 10(-9) M concentration became effective after 5 h of incubation. The E2 effects involved the estrogen receptor (ER) alpha because they were similarly obtained with the specific ER alpha agonist 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole. The use of glutamate receptor agonists and antagonists indicated that E2 effects on GnRH secretion evoked by both glutamate and L-arginine involved the 2-amino-3-hydroxy-5-methyl-4-isoxazol propionic acid/kainate receptors. Similar E2 effects on the kainate-evoked secretion were observed throughout development in both sexes. The observation of similar E2 effects using explants containing the median eminence alone indicated that the median eminence was a direct target for E2 rapid effects on the glutamate-evoked GnRH secretion. The signaling pathways involved in E2 effects included an increase in intracellular calcium and the activation of protein kinase A, protein kinase C, and MAPK. It is concluded that E2 can stimulate the glutamate- and nitric oxide-evoked GnRH secretion in vitro through a rapid pathway involving the ER and kainate receptor as well as through a slower mechanism responding to lower E2 concentrations.

Prevalence and demographic features of childhood growth hormone deficiency in Belgium during the period 1986-2001.

OBJECTIVE: Since the availability of recombinant human growth hormone (rhGH) all children with growth hormone deficiency (GHD) living in Belgium are offered rhGH treatment after approval by a peer-review board. In this study, we evaluated the prevalence and demographic features of childhood GHD in Belgium during the period 1986-2001 and we compared them with the data from other countries. METHODS: Diagnostic, demographic and baseline auxological data of 714 children diagnosed as having GHD between 1986 and 2001 were retrieved from the database of the Belgian Study Group for Paediatric Endocrinology. RESULTS: The prevalence of GHD in Belgium was estimated to be 1/5600. The origin of GHD was idiopathic (idGHD) in 41% of the patients, congenital (congGHD) in 20% and acquired (acqGHD) in 35%. During the first 4 years (1986-1989) more patients were classified as idGHD; thereafter the distribution between the three aetiology groups did not change. In all groups, boys outnumbered girls but this preponderance was especially pronounced in congGHD patients (male:female=4:1) with a central malformation that associates an anterior pituitary hypoplasia, a missing, fine or normal pituitary stalk and an ectopic posterior pituitary. Thirteen percent of the patients with idGHD, 50% with congGHD and 52% with acqGHD had multiple pituitary deficiencies. Patients with congGHD were the youngest (mean+/-s.d. age: 6.5+/-4.7 years) and were the shortest (-3.0+/-1.3 standard deviation score (SDS)) at the start of rhGH treatment. There was no time trend over the studied period for age and height at onset of GH therapy. CONCLUSION: In Belgium, the prevalence of childhood GHD can be estimated as 1/5600 which is comparable to other recent surveys. The yearly number of new patients for the different aetiologies and the auxological parameters have remained relatively constant over the last 16 years.

Estradiol stimulation of pulsatile gonadotropin-releasing hormone secretion in vitro: correlation with perinatal exposure to sex steroids and induction of sexual precocity in vivo.

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.

Induction of puberty in the hypogonadal girl--practices and attitudes of pediatric endocrinologists in Europe.

The management of children and adolescents with hypogonadism and in particular the induction of puberty in the hypogonadal girl is subject to controversy. Therefore, under the auspices and through organization of the Drugs and Therapeutics Committee of the European Society of Paediatric Endocrinology (ESPE), an interactive voting session and workshop was held at the 39th ESPE Annual Meeting in Brussels to discuss these topics. Common practice in Europe and attitudes of pediatric endocrinologists in Europe were questioned and recorded in the 1.5-hour program. We now report on some of the results of the questionnaires and discussions of that session to further the discussion on and knowledge of current concepts of induction of puberty in the hypogonadal girl in Europe. It became clear from the data accumulated here that the start of treatment, the aims of therapy and the modalities of how to treat the hypogonadal girl vary amongst pediatric endocrinologists in Europe. For example, a chronological age > or =11 years was considered appropriate for the start of estrogen therapy by 40.4% (out of 188 answers), while 47.8 and 7.5% felt that a chronological age > or =13 and > or =15 years respectively was appropriate. In respect to the form and route of estrogen administration, the audience was asked for their common estrogen replacement practice: 31.9% used oral 17beta-estradiol treatment, while 10% would prescribe 17beta-estradiol transdermal patches. Another 12.2% would recommend conjugated estrogens (e.g. Premarin) orally, 4.8% use oral estradiol valerate and 39.3% ethinylestradiol orally. Only 1.8% out of 229 physicians answering were undecided. In addition, counseling of patients and their families is quite variable and perceptions for example regarding potential pregnancies in affected women are also not uniform. In this report the authors do not want to provide their own personal views but rather reflect current practice in Europe. It is hoped that a more uniform picture will emerge once European and international guidelines on how to treat the girl with hypogonadism will be available and even more discussions amongst doctors from different countries have been led.

Sexual precocity after immigration from developing countries to Belgium: evidence of previous exposure to organochlorine pesticides.

In a retrospective auxological study of 145 patients seen in Belgium during a 9-year period for treatment of precocious puberty, 28% appeared to be foreign children (39 girls, one boy) who immigrated 4 to 5 years earlier from 22 developing countries, without any link to a particular ethnic or country background. The patients were either adopted (n = 28) or non-adopted (n = 12), the latter having normal weight and height at immigration and starting early puberty without evidence of earlier deprivation. This led to the hypothesis that the mechanism of precocious puberty might involve previous exposure to oestrogenic endocrine disrupters. A toxicological plasma screening for eight pesticides detected p,p'-DDE, which is derived from the organochlorine pesticide DDT. Median p,p'-DDE concentrations were respectively 1.20 and 1.04 ng/ml in foreign adopted (n = 15) and non-adopted (n = 11) girls with precocious puberty, while 13 out of 15 Belgian native girls with idiopathic or organic precocious puberty showed undetectable concentrations (<0.1 ng/ml). A possible relationship between transient exposure to endocrine disrupters and sexual precocity is suggested, and deserves further studies in immigrant children with non-advanced puberty.

Leptin effects on pulsatile gonadotropin releasing hormone secretion from the adult rat hypothalamus and interaction with cocaine and amphetamine regulated transcript peptide and neuropeptide Y.

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 increased by leptin (10(-7) M) and CART (10(-6) 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 amplitude in the female. A slight but significant increase in GnRH pulse amplitude was caused by NPY (10(-7) M) in the female. However, GnRH pulse amplitude was not affected by a Y5-receptor antagonist (10(-6) 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 anti-CART antiserum whereas it was not affected by coincubation with the NPY Y5-receptor antagonist (10(-7) 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.

Management of central isosexual precocity: diagnosis, treatment, outcome.

The diagnosis of central isosexual precocity, a condition much more common in girls than in boys, is currently viewed as a spectrum of disorders between isolated premature thelarche and borderline early puberty. In some countries, a trend may be seen toward onset of puberty at earlier ages. Integration of the clinical findings with bone age, pelvic echography, and hormonal data as well as follow-up ascertainment of progression of development is critical to define which patients should be proposed for therapy. The use of long-acting forms of gonadotropin-releasing hormone (GnRH) agonists may not be indicated in slowly progressive variants or borderline early puberty because they do not affect final height. Preservation of height potential is particularly obvious in precocious puberty starting at young ages. In some selected patients, associated growth hormone therapy may increase adult height but further studies are warranted. The psychosocial and behavioral correlates of precocious puberty are an important and under investigated area.

Cocaine and amphetamine-regulated-transcript peptide mediation of leptin stimulatory effect on the rat gonadotropin-releasing hormone pulse generator in vitro.

Pulsatile gonadotropin-releasing hormone (GnRH) secretion was studied in vitro using explants of the retrochiasmatic hypothalamus from prepubertal male and female rats. Leptin caused a dose-dependent reduction of the GnRH interpulse interval in both sexes. We studied the effects of cocaine- and amphetamine-regulated transcript (CART) since this peptide was shown recently to mediate the anorectic effects of leptin in the hypothalamus. CART caused a reduction of the GnRH interpulse interval. This effect was prevented using an anti-CART antiserum which could partially overcome leptin stimulatory effects as well. Using hypothalamic explants from Zucker rats homozygous for the leptin receptor mutation ( fa/fa), GnRH pulse frequency was not affected by leptin, while a significant acceleration was caused by the CART-peptide. In conclusion, leptin involves the hypothalamic CART-peptide to stimulate the prepubertal GnRH pulse generator in vitro.

In vitro stimulation of the prepubertal rat gonadotropin-releasing hormone pulse generator by leptin and neuropeptide Y through distinct mechanisms.

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.

Early prepubertal ontogeny of pulsatile gonadotropin-releasing hormone (GnRH) secretion: I. Inhibitory autofeedback control through prolyl endopeptidase degradation of GnRH.

GnRH[1-5], a subproduct resulting from degradation of GnRH by prolyl endopeptidase (PEP) and endopeptidase 24.15 (EP24.15) was known to account for an inhibitory autofeedback of GnRH secretion through an effect at the N-methyl-D-aspartate (NMDA) receptors. This study aimed at determining the possible role of such a mechanism in the early developmental changes in frequency of pulsatile GnRH secretion. Using retrochiasmatic explants from fetal male rats (day 20-21 of gestation), no GnRH pulses could be observed in vitro, whereas pulses occurred at a mean interval of 86 min from the day of birth onwards. This interval decreased steadily until day 25 (39 min), during the period preceding the onset of puberty. Based on GnRH[1-10] or GnRH[1-9] degradation and GnRH[1-5] generation after incubation with hypothalamic extracts, EP24.15 activity did not change with age, whereas PEP activity was maximal at days 5-10 and decreased subsequently until day 50. These changes were consistent with the ontogenetic variations in PEP messenger RNAs (mRNAs) quantitated using RT-PCR. Using fetal explants, the NMDA-evoked release of GnRH was potentiated in a dose-dependent manner by bacitracin, a competitive PEP inhibitor and the desensitization to the NMDA effect was prevented using 2 mM of bacitracin. At day 5, a higher bacitracin concentration of 20 mM was required for a similar effect. Pulsatile GnRH secretion from fetal explants was not caused to occur using bacitracin or Fmoc-Prolyl-Pyrrolidine-2-nitrile (Fmoc-Pro-PyrrCN), a noncompetitive PEP inhibitor. At postnatal days 5 and 15, a significant acceleration of pulsatility was obtained using 1 microM of Fmoc-Pro-PyrrCN or 2 mM of bacitracin. At 25 and 50 days, a lower bacitracin concentration of 20 microM was effective as well in increasing the frequency of GnRH pulsatility. We conclude that the GnRH inhibitory autofeedback resulting from degradation of the peptide is operational in the fetal hypothalamus but does not explain the absence of pulsatile GnRH secretion at that early age. After birth, PEP activity is high and may account for the low frequency of pulsatility. The potency of that effect decreases before the onset of puberty and may contribute to the acceleration of GnRH pulsatility.

[The physician faced with an iodine deficiency: individual management of a public health problem]. / Le praticien face à la carence iodée: prise en charge individuelle d'un problème de santé publique.

Urinary iodine was measured in samples collected during the first week of life in newborns from the areas of Liège in Belgium and Cluj in Romania. In Liège, severe iodine deficiency was seen in 1 out of 8 newborns and mild iodine deficiency in 1 out of 3. A greater proportion of newborns showed iodine deficiency in Cluj. Since public health measures to prevent iodine deficiency have not been set up yet by belgian authorities, the practitioner has to ensure individually optimal iodine intake, particularly in pregnant women, in newborns and during infancy.

[Programs of systematic screening in neonatology. Pharmaco-economic aspects]. / Les programmes de dépistage systématique en néonatologie. Aspects pharmaco-économiques.

Systematic neonatal screening offers many advantages for the patients, its family and the community. The Genetic center of the University Hospital of Liège provides neonatal screening for the following diseases: phenylketonuria, congenital hypothyroidy, cystic fibrosis, alpha-1-antitrypsin, adrenal hyperplasia and biotinidase deficiency. On economical grounds, it is clear that the organisation of neonatal screening costs less to the community than the cost of the disease if diagnosis is made too late as to allow an alleviation, or even a total recovery, of the symptomatology.

Pulsatile secretion of gonadotropin-releasing hormone by rat hypothalamic explants without cell bodies of GnRH neurons [corrected].

Gonadotropin-releasing hormone (GnRH) is typically secreted in a pulsatile manner. It is still unclear whether pulsatility depends on a GnRH pulse generator residing in the GnRH neurons or in other neurons. Since the cell bodies of GnRH neurons are located rostrally to the optic chiasm and the majority of GnRH terminals in the median eminence of the rat hypothalamus, we have compared GnRH secretion from individual preoptic, retrochiasmatic and median eminence explants using a static incubation system. GnRH is released from the three different types of explant in response to depolarization with veratridine or glutamate receptor stimulation using the agonist N-methyl-D-aspartate. Only the retrochiasmatic explants, however, show a characteristic pulsatile secretion of GnRH. The mean (+/- SD) interpulse interval is respectively 37 +/- 5, 25 +/- 4 and 12 +/- 1 min when the fractions are collected at 7.5-, 5.0- and 2.5-min intervals. The immunocytochemically stained GnRH cell bodies are normally distributed in the preoptic explants (n = 212-420) while only 3 GnRH cell bodies can be visualized in 7 retrochiasmatic explants. Semi-quantitative RT-PCR shows that GnRH mRNA is present in the retrochiasmatic explant in a ratio of about 1:600 relative to the preoptic explant. We conclude that pulsatile GnRH secretion occurs in the virtual absence of GnRH cell bodies but does not occur from GnRH terminals in the isolated median eminence. These data further indicate that a mechanism of GnRH pulsatility is located in the retrochiasmatic hypothalamus and involves neurons different from the GnRH neurons.

Amino acid neurotransmission and initiation of puberty: evidence from nonketotic hyperglycinemia in a female infant and gonadotropin-releasing hormone secretion by rat hypothalamic explants.

The pulse frequency of hypothalamic GnRH secretion increases at the onset of puberty. In rodents and primates, this process involves facilitatory and inhibitory effects mediated through hypothalamic N-methyl-D-aspartic acid (NMDA) and gamma-aminobutyric acid (GABA) receptors, respectively. Precocious puberty was observed in an 11-month-old girl with nonketotic hyperglycinemia. This was thought to result from the effect of high concentrations of glycine (112 micromol/L in cerebrospinal fluid; normal, 3-12) acting on NMDA receptors as a coagonist of glutamate. Regression of pubertal development during anticonvulsive treatment with GABA agonists (loreclezole and vigabatrin) suggested that the stimulatory effects of glycine could be overcome by GABA receptor-mediated inhibition. These two hypotheses were tested in the in vitro model of the explanted hypothalamus from infantile (15-day-old) male rats. Glycine concentrations of 1-10 micromol/L increased the pulse frequency of GnRH secretion. This acceleration was prevented by 7-chlorokynurenic acid, a glycine antagonist at the NMDA receptor complex, and by the GABA agonist loreclezole. In addition, loreclezole and vigabatrin suppressed the developmental increase in the frequency of pulsatile GnRH secretion. The observation of precocious puberty in an infant with hyperglycinemia followed by pubertal regression during GABA agonist therapy and the in vitro findings in hypothalamic explants suggest that stimulatory inputs mediated through NMDA receptors and inhibitory inputs through GABA receptors are involved in the initiation of puberty.

Duality of glutamatergic and GABAergic control of pulsatile GnRH secretion by rat hypothalamic explants: I. Effects of antisense oligodeoxynucleotides using explants including or excluding the preoptic area.

Using antisense oligodeoxynucleotides we aimed to study the role of N-methyl-D-aspartate (NMDA) and gamma-aminobutyric acid (GABA) receptors in the mechanism of Gonadotrophin-releasing hormone (GnRH) secretion in vitro. Since GnRH cell bodies are located in the rat preoptic hypothalamus while most GnRH terminals are in the retrochiasmatic hypothalamus, we compared the effects of oligodeoxynucleotides on explants of the whole (preoptic area included) or retrochiasmatic hypothalamus. When GnRH secretion is evoked by muscimol and NMDA, a time-related reduction of GnRH secretion is caused by antisense oligodeoxynucleotides for the beta subunit of the GABAA receptor and the NR2A subunit of the NMDA receptor, respectively. After 6-7 h, binding studies of tritiated ligands show a decrease in GABA- and NMDA-receptor expression. While these antisense effects are observed using whole explants, no such effects are seen using retrochiasmatic explants, indicating that the facilitatory GABAA and NMDA receptors are encoded in the preoptic area. Using several missense oligodeoxynucleotides or antisense for the NR2B and NR2C subunits of the NMDA receptor, the muscimol- and NMDA-evoked release of GnRH is not affected. When spontaneous pulsatile GnRH secretion is studied, the NR2A antisense oligodeoxynucleotides cause an increase of the interpulse interval. This increase is seen using whole but not retrochiasmatic explants. In contrast, the GABAA and NR2C antisense oligodeoxynucleotides result in a reduction of GnRH interpulse interval. Such a reduction is seen using whole as well as retrochiasmatic explants, indicating that the GABAA and NMDA receptors which mediate inhibition of GnRH pulsatility are encoded in the retrochiasmatic hypothalamus. We conclude that NMDA receptors (NR2A subunit) encoded in the preoptic hypothalamus mediate a facilitatory effect on GnRH pulsatility while GABAA and NMDA (NR2C subunit) receptors encoded in the retrochiasmatic hypothalamus mediate an inhibition of GnRH pulsatility. Pulsatile GnRH secretion is affected differently than the agonist-evoked release of GnRH suggesting that the GnRH secretory neurons and the GnRH pulse generator consist of different cellular entities.