GnRH Synthesis Research Articles

IMPACT OF RETINAL DEGENERATION ON MELATONIN AND GONADOTROPIN INHIBITORY HORMONE LEVELS DURING PHOTOSTIMULATION IN SMOKY JOE CHICKENS

Event Abstract Back to Event IMPACT OF RETINAL DEGENERATION ON MELATONIN AND GONADOTROPIN INHIBITORY HORMONE LEVELS DURING PHOTOSTIMULATION IN SMOKY JOE CHICKENS Gregoy Y. Bedecarrats1*, Jason Fleming1, Takayoshi Ubuka2, Yoshihisa Hasegawa3 and Kazuyoshi Tsutsui2 1 University of Guelph, Animal and Poultry Science, Canada 2 Waseda University, Biology, Japan 3 Kitasato University, Reproductive Endocrinology, Japan In avian species the reproductive axis is controlled by stimulatory (GnRH) and inhibitory (GnIH) inputs. Light stimulates the synthesis and release of GnRH via hypothalamic photoreceptors, and indirectly controls GnIH production by altering melatonin (Mel) synthesis from the retina and pineal gland. Recently, we reported that blind roosters from the Smoky Joe strain of chickens mature sexually earlier than sighted animals. Thus, in the present study we investigated if the lack of functional retina impacts Mel and GnIH levels. Smoky Joe roosters were maintained on a short-day photoperiod (8h/SDP) until 18 wks of age then transferred to a long-day photoperiod (14h/LDP). At 18 (SDP) and 24 (LDP) wks of age, blood samples were collected every 4h for 24h from 10 blind and 10 sighted birds. Additionally, for both collection ages, pineal, retina and diencephalons were collected from 5 blind and 5 sighted birds in the middle of the dark and light phases. In blind birds, retinal Mel levels were low in all samples while in sighted birds, they were significantly increased during the dark phase under SDP but not LDP. Pineal Mel content was significantly higher during the dark than the light phase for both blind and sighted animals under SDP. Interestingly, under LDP, pineal Mel content in sighted roosters was significantly lower than those observed under SDP. Despite the difference observed in the retina and pineal, plasma Mel profiles were identical between blind and sighted birds with a significant increase during dark phases under both SDP and LDP. Levels of GnIH in the diencephalon mirrored those observed for Mel in the pineal gland with decreased levels during light phases in both blind and sighted birds, and lower levels during dark phase in sighted roosters under LDP. Our data indicate that although Mel and GnIH follow a circadian rhythm in both blind and sighted roosters, a functional retina results in decreased pineal Mel and hypothalamic GnIH during LDP dark phases. Acknowledgements This work was supported in part by the Poultry Industry Council, the Natural Sciences and Engineering Research Council of Canada, and the Ontario Ministry of Agriculture, Food and Rural Affairs. Keywords: chicken, GnIH, Melatonin, Reproduction Conference: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology, Ann Arbor, United States, 13 Jul - 16 Jul, 2011. Presentation Type: Poster Topic: Neuroendocrine control of reproduction Citation: Bedecarrats GY, Fleming J, Ubuka T, Hasegawa Y and Tsutsui K (2011). IMPACT OF RETINAL DEGENERATION ON MELATONIN AND GONADOTROPIN INHIBITORY HORMONE LEVELS DURING PHOTOSTIMULATION IN SMOKY JOE CHICKENS. Front. Endocrinol. Conference Abstract: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology. doi: 10.3389/conf.fendo.2011.04.00004 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 11 Jul 2011; Published Online: 09 Aug 2011. * Correspondence: Dr. Gregoy Y Bedecarrats, University of Guelph, Animal and Poultry Science, Guelph, N1G 2W1, Canada, gbedecar@uoguelph.ca Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Gregoy Y Bedecarrats Jason Fleming Takayoshi Ubuka Yoshihisa Hasegawa Kazuyoshi Tsutsui Google Gregoy Y Bedecarrats Jason Fleming Takayoshi Ubuka Yoshihisa Hasegawa Kazuyoshi Tsutsui Google Scholar Gregoy Y Bedecarrats Jason Fleming Takayoshi Ubuka Yoshihisa Hasegawa Kazuyoshi Tsutsui PubMed Gregoy Y Bedecarrats Jason Fleming Takayoshi Ubuka Yoshihisa Hasegawa Kazuyoshi Tsutsui Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Neurokinin B and Kisspeptin: Sexual Partners or Single Agents?

Despite recent progress in our understanding of reproductive physiology, the precise mechanisms controlling puberty still remainelusive.Patientswith idiopathichypogonadotrophic hypogonadism (IHH) have pubertal failure as a result of insufficient gonadotrophin release, which is usually caused by a defect in GnRH synthesis, secretion, or activity. Inactivatingmutations inanumberofgeneshavebeen found to cause IHH, but the underlying genetic defect in many patients with IHH remains unidentified (1). Two novel neuropeptides, kisspeptin and neurokinin B, have emerged recently as “new kids on the block” and caused an explosion of research in reproductive endocrinology. Their similar anatomical and functional characteristics have prompted speculation whether a “sexual partnership” exists between them to control reproductive maturation. Kisspeptin emerged as an important new player in reproductivephysiology in2003whendeRouxetal. (2)andSeminara et al. (3) discovered that humans with inactivating mutations of the kisspeptin receptor had a phenotype of hypogonadotrophic hypogonadism. Furthermore, animals lacking kisspeptin (4, 5) or its receptor (3) have a similar phenotype. Over the past 7 yr, more than 400 subsequent publications have elucidated the effects of kisspeptin (comprehensively reviewed in Ref. 6). Notably, administration of kisspeptin potently stimulates reproductive hormone release in all species studied (6), including humans (7–9). The stimulatory action of kisspeptin on gonadotrophin release is abolished by pharmacological antagonism of GnRH, thus suggesting a hypothalamic site of action (6). Kisspeptin is expressed in specific hypothalamic nuclei, such as the anteroventral periventricular and arcuate nuclei, which each have important roles in its physiological action (6). Recently, it has emerged that neurokinin B is also vital for normal pubertal function. Topaloglu et al. (10) discovered that inactivating mutations of neurokinin B or its receptor caused hypogonadotrophic hypogonadism in humans. Recent data show that neurokinin B and kisspeptin are colocalized in the arcuate nucleus in sheep (11), mice (12), rats (13), and humans (14). However, in contrast to the robust stimulatory action of kisspeptin on gonadotrophin release demonstrated in numerous species, the effects of administering neurokinin B to animals are less clear. Neurokinin B administration has been shown stimulate LH release in sheep (15) but have no effect (16, 17) or inhibit (12, 18) LH release in rodents. It is also interesting that mice with targeted deletion of neurokinin B receptor are fertile (19), in contrast to kisspeptin or kisspeptin receptor knockout mice (3–5). It is therefore timely that, in this issue of Endocrinology, Ramaswamy et al. (20) demonstrate in elegant primate studies that a single i.v. injection of neurokinin B robustly stimulatesLHrelease.Thiswork follows fromstudiesofkisspeptin administration to primates, performed by the same researchers. As observed previously with kisspeptin administration (21), the effect of neurokinin B on LH release was abolished by GnRH receptor antagonism (20), thus suggesting a hypothalamic site of action. Ramaswamy et al. also show that neurokinin B and kisspeptin are colocalized in the arcuate nucleus of primates (20), which is consistent with the findings inrodents, sheep,andhumans(11–14).However, in contrast to previous results with kisspeptin (22), repeated hourly injection of a neurokinin B agonist did not result in sustainedLHrelease(20). Interestingly,repeatedadministrationof the neurokinin B did not affect the ability of kisspeptin to stimulate GnRH release (20). The precise animal model chosen by Ramaswamy et al. in the current study may be important; ago-

Reversible Isolated Hypogonadotropic Hypogonadism due to Mutations in the Neurokinin B Regulation of Gonadotropin-Releasing Hormone Release

In this commentary, congenital isolated hypogonadotropic hypogonadism (IHH) is defined as absent, incomplete, or arrested isosexual development when both chronological and bone ages are at least 18 yr, together with low concentrations of gonadotropins and sex hormones in the absence of systemic disease, syndromic malformations, nutritional deprivation, and other functional or anatomic pituitary abnormalities. To date, the identified forms of IHH are primarily due to inactivating mutations in the genes whose products control: 1) the differentiation and development (NROB1 or DAX1, CHD7, FGFR1, FGF8); 2) the subsequent migration of neurons that synthesize and secrete GnRH from their embryological site of origin in the nasal (olfactory) placode to their network in the hypothalamic arcuate nucleus (infundibulum); 3) the synthesis, release, and action of GnRH; and 4) the synthesis and secretion of the gonadotropins (LH, FSH) (Table 1) (1, 2). The migration of GnRH-synthesizing nerve cells requires the coordinated activity of the products encoded by KAL1, FGFR1, FGF8, NELF, PROK2, PROKR2, and likely many other factors yet to be identified. Inasmuch as loss-of-function mutations in these genes are often associated with abnormalities of olfaction (anosmia, hyposmia), they constitute the defined forms of Kallmann syndrome. Disrupted expression of factors (LEP, LEPR, KISS1R) that regulate the synthesis of GnRH (GNRH1), its storage in the median eminence, its release into the hypophyseal-portal vasculature, its action upon the pituitary gonadotrope (GHAHR), or the expression and synthesis of the gonadotropins (LH , FSH , PCSK1) themselves also leads to IHH (3). To the list of modulators of GnRH synthesis and secretion have recently been added the products of TAC3 (tachykinin 3 or neurokinin B) and TACR3 (the G proteincoupled receptor for tachykinin 3) (4, 5). Gianetti et al. (6) have now genotyped TAC3 and TACR3 in 345 patients (247 males) with IHH and have identified both nonsense and missense nonsynonymous and synonymous mutations in TACR3 in 19 patients (four females) (5.5%) as well as a frameshift mutation in TAC3 in one woman (0.3%). In nine subjects, the TACR3 mutation was biallelic and in eight of nine homozygotic, whereas in 10 patients the mutations in TACR3 were monoallelic. Both recessive and dominant modes of IHH transmission were present in patients with mutations in TACR3. Patients with heterozygous mutations in TACR3 were clinically similar to the subjects with homozygous mutations. In none of the patients with heterozygous mutations in TACR3 were mutations in other examined IHH genes identified, suggesting that digenic transmission of IHH was less likely in these subjects (7). Functional characterization of several of the missense mutations in TACR3 revealed that the DNA base changes inactivated the product except for two monoallelic mutations (Gly18Asp and Ile249Val) in which in vitro bioactivity was normal, leaving unexplained the mechanism by which these mutations resulted in IHH in their carriers. Clinically, 14 of 15 males with IHH due to mutations in TACR3 had phallic lengths that were less than 10.5 cm and were classified as micropenis. As anticipated, at diagnosis of IHH, most affected males had very small testes, but in some patients testicular volumes were a bit larger than prepubertal, a size increase attributable to endogenous mechanisms. After discontinuation of sex hormone replacement therapy, testicular volume and/or gonadotropin and/or testosterone secretion increasedspontaneously infivemales, twoofwhomreported

Converse Regulatory Functions of Estrogen Receptor-α and -β Subtypes Expressed in Hypothalamic Gonadotropin-Releasing Hormone Neurons

Estradiol (E(2)) acts as a potent feedback molecule between the ovary and hypothalamic GnRH neurons, and exerts both positive and negative regulatory actions on GnRH synthesis and secretion. However, the extent to which these actions are mediated by estrogen receptors (ERs) expressed in GnRH neurons has been controversial. In this study, Single-cell RT-PCR revealed the expression of both ERalpha and ERbeta isoforms in cultured fetal and adult rat hypothalamic GnRH neurons. Both ERalpha and ERbeta or individual ERs were expressed in 94% of cultured fetal GnRH neurons. In adult female rats at diestrus, 68% of GnRH neurons expressed ERs, followed by 54% in estrus and 19% in proestrus. Expression of individual ERs was found in 24% of adult male GnRH neurons. ERalpha exerted marked G(i)-mediated inhibitory effects on spontaneous action potential (AP) firing, cAMP production, and pulsatile GnRH secretion, indicating its capacity for negative regulation of GnRH neuronal function. In contrast, increased E(2) concentration and ERbeta agonists increase the rate of AP firing, GnRH secretion, and cAMP production, consistent with ERbeta-dependent positive regulation of GnRH secretion. Consonant with the coupling of ERalpha to pertussis toxin-sensitive G(i/o) proteins, E(2) also activates G protein-activated inwardly rectifying potassium channels, decreasing membrane excitability and slowing the firing of spontaneous APs in hypothalamic GnRH neurons. These findings demonstrate that the dual actions of E(2) on GnRH neuronal membrane excitability, cAMP production, and GnRH secretion are mediated by the dose-dependent activation of ERalpha and ERbeta expressed in hypothalamic GnRH neurons.

Courtship interactions stimulate rapid changes in GnRH synthesis in male ring doves

Many birds and mammals show changes in the hypothalamo-pituitary-gonadal (HPG) axis in response to social or sexual interactions between breeding partners. While alterations in GnRH neuronal activity play an important role in stimulating these changes, it remains unclear if acute behaviorally-induced alterations in GnRH release are accompanied by parallel changes in GnRH synthesis. To investigate this relationship, we examined changes in the activity of GnRH neurons in the brains of male ring doves following brief periods of courtship interactions with females. Such interactions have been previously shown to increase plasma LH in courting male doves at 24 h, but not at 1 h, after pairing with females. In the first study, males allowed to court females for 2 h had 60% more cells that showed immunocytochemical labeling for GnRH-I in the preoptic area (POA) of the hypothalamus than did control males that remained isolated from females. To determine whether an increase in GnRH gene expression preceded this increase in GnRH immunoreactivity in the POA, changes in the number of cells with detectable GnRH-I mRNA in the POA were measured by in situ hybridization following a 1 h period of courtship interactions with females. In this second study, courting males exhibited 40% more cells with GnRH-I in this region than did isolated control males. GnRH-immunoreactive neurons in two other diencephalic regions failed to show these courtship-induced changes. Plasma LH was not elevated after 1 or 2 h of courtship. These results demonstrate that the release of GnRH-I in the POA that is presumably responsible for courtship-induced pituitary and gonadal activation is accompanied by a rapid increase in GnRH synthesis that occurs before plasma LH levels increase. We suggest that this increase in GnRH synthesis is necessary to support the extended period of HPG axis activation that is seen in this species during the 5–10 day period of courtship and nest building activity.

Central Application of IGF-1 Postpones Time of Vaginal Opening in Normally Fed, but Not in Food-Restricted Rats

Background/Aims: Central but also peripheral IGF-1 is suggested to play a role in the initiation of puberty as it directly affects GnRH synthesis and release. A possible intermediate in the effects of IGF-1 on puberty might be the adiposity-signaling hormone leptin, whose plasma levels are decreased in food-restricted (FR) rats. Methods: IGF-1 was chronically centrally infused in 23-day-old prepubertal female rats which were either normally fed or 30% FR, and the effects on time of vaginal opening (VO) and plasma leptin levels were monitored. Results: FR treatment postponed time of VO and decreased plasma leptin levels. In normally fed rats centrally infused with IGF-1, time of VO was found to be postponed to the same extent as FR treatment did. The IGF-1 infusion did not affect plasma leptin levels in normally fed animals but increased leptin levels in the FR group compared to controls. Daily food intake was equal between all groups but body weight course was lower in FR rats. IGF-1 treatment did not significantly affect food intake or body weight course. Conclusion: FR treatment delays the moment of vaginal opening to the same extent as observed in normally fed rats that were centrally infused with IGF-1.

Role of bio-genic amines for the hypothalamic regulation of reproductive function

The present review is dedicated to monoaminergic systems involved in hypothalamic regulation of reproduction, as well as to the role of the systems in GnRH synthesis and secretion regulation. Data are presented on the ovarian steroid hormones, the suprachiasmatic nuclei of the hypothalamus, in which the central oscillator of a mammalian organism's diurnal rhythms is located, and on the pineal hormone melatonin involved in formation of reproductive cycles. Both own results and literature data referring to the role of diurnal rhythms of the activity of monoaminergic systems of the hypothalamus in the central regulation of reproduction are discussed.

Impaired hypothalamic regulation of reproductive function when exposed to neurotoxic compounds and melatonin

Diurnal rhythms of the contents of biogenic amines in the hypothalamus are considered to play an important role in regulation of many functions such as motor activity, feeding behaviour, sleep-awake cycle formation as well as hormonal and immune statuses. Mechanisms of regulation of reproduction that participate in a circadian signal transmission from the suprachiasmatic nuclei (SCN) of the hypothalamus to hypothalamic structures responsible for gonadoliberin (GnRH) synthesis and secretion (the preoptic area - PA, and the medial eminence - ME) are at present being intensively studied. It has been shown that the aging caused disturbances of estrous cycles in female rats are accompanied by a disappearance of diurnal rhythms of activity of monoaminergic and opioid hypothalamic systems playing a key role in regulation of GnRH synthesis and secretion.
 The experiments carried out have revealed diurnal rhythms of the contents of the studied biogenic amines in the PA, ME and SCN of the hypothalamus. The rhythms are due to the amines increasing in the morning hours when compared to their evening and night levels. The neurotoxic compounds used disturbed the rhythms of the amines. Chronic inhalation of toluene caused a disappearance of the dynamics of all the studied neurotransmitters that was typical of the control group. Meanwhile, a single administration of 1,2-dimethylhydrazine resulted in either a complete disappearance of the diurnal rhythms of the biogenic amines contents found in control, or their phase shifts. Severe disturbances of the GnRH content and of the ROS diurnal dynamics were also observed in the studied hypothalamic areas.
 The literature allowed supposition that melatonin possessing entraining and antioxidant properties can be used as a protector of disturbances of central mechanisms of regulation of reproduction. However, our experiments have shown that exogenous melatonin does not only eliminate the neurotoxic compounds caused disturbances of diurnal rhythms of the studied indices, but it itself causes deep changes in their dynamics. The effect of melatonin has been found to result in a significant decrease of the catecholamines contents in all the studied hypothalamic structures, particularly of the norepinephrine content, that is accompanied by a profound decrease of the GnRH content and by a disturbance of the diurnal dynamics of reactive oxygen species (ROS) in the PA and ME. The effect of 1,2-dimethylhydrazine on the studied indices was less pronounced when compared to that of melatonin, whereas an injection of the xenobiotic on the background of the pineal gland hormone administration resulted in a predominant effect of the latter.
 Both the data obtained and the literature review allow to offer a hypothesis for a possible mechanism of involvement of diurnal rhythms of biogenic amines in central regulation of reproduction. It is known that synthesis and secretion of endogenous opioid peptides in the mediobasal hypothalamus undergo diurnal oscillations with minimum in the morning and maximum in the evening and/or at night. The revealed diurnal rhythms of the biogenic amines contents had opposite oscillations. This fact allows a supposition that these rhythms are due to the opioid system activity changing. Both decreased endogenous opioids contents and simultaneously increased morning biogenic amines levels in proestrus can create a neurotransmitter background that is necessary for GnRH surge formation.
 An evaluation of diurnal rhythms of the biogenic amines contents, GnRH and ROS in the studied hypothalamic structures can be used for an assessment of disturbances of central mechanisms of regulation of reproduction that are caused by other neurotoxic compounds or developed in some diseases and in aging when the functional activity of the pineal gland is altered. The data obtained testify to possible negative side effects of melatonin on hypothalamic regulation of reproduction in therapeutic use of the hormone. The effects of melatonin realized via its receptors are known to highly depend on amplitude of its nocturnal surge and on duration of its increased blood level during a day. Therefore administration of melatonin on the background of its normal synthesis can lead to a number of negative effects due to its increased blood level when compared to its physiological level. Elucidation of the exogenous melatonin caused disturbance of the studied diurnal rhythms of central regulation of reproduction is of great practical use in development of prophylactics helping to eliminate or decrease an effect of damaging factors in unfavourable environmental conditions.

Aging-Related Increased Expression of Inducible Nitric Oxide Synthase and Cytotoxicity Markers in Rat Hypothalamic Regions Associated with Male Reproductive Function

We have previously demonstrated that the inducible nitric oxide synthase (iNOS) protein and total NOS activity increase in the hypothalamus and other regions of the male rat brain during aging. We have now tested the hypothesis that increased iNOS results in excessive nitric oxide (NO) and peroxynitrite production, and leads to increased apoptosis in CNS cells, including the GnRH and oxytocin hypothalamic neurons involved in the control of male reproductive function. Young (3-month-old) and old (24-month-old) male Brown Norway rats (n = 6) were perfused with 4% formalin. Adjacent coronal paraffin-embedded sections (5 µm) of preoptic area (POA), supraoptic nucleus (SON), paraventricular nucleus (PVN), and arcuate nucleus (ARC) of the hypothalamus were immunostained with antibodies for iNOS, neuronal NOS (nNOS), and nitrotyrosine (a marker of peroxynitrite formation). The intensity of immunostaining was measured using a densitometric image analysis system. Apoptosis was determined by the TUNEL assay. Double immunofluorescence staining with confocal laser scanning microscopy was used for co-localization studies. A significant increase in the iNOS immunostaining measured as optical density (OD) was found in the old compared to the young animals (SON: 0.32 ± 0.02 vs. 0.23 ± 0.03, p < 0.05; PVN: 0.34 ± 0.03 vs. 0.07 ± 0.05, p < 0.001; POA: 0.18 ± 0.02 vs. 0.01 ± 0.02, p < 0.001). Aging did not affect nNOS expression. Nitrotyrosine was elevated in the hypothalamic regions of old compared to young rats (SON: 0.32 ± 0.05 vs. 0.10 ± 0.04, p < 0.05; PVN: 0.32 ± 0.04 vs. 0.13 ± 0.03, p < 0.01; POA: 0.72 ± 0.06 vs. 0.03 ± 0.003, p < 0.001). Increased nitrotyrosine was accompanied by an elevation of the apoptotic index in the old rats (SON: 11.01 ± 3.33 vs. 0.57 ± 0.50, p < 0.001; PVN: 3.08 ± 1.12 vs. 0.42 ± 0.32; POA: 6.60 ± 1.93 vs. 0.18 ± 0.17, p < 0.01; ARC: 0.001 ± 0.0001 vs. 4.33 ± 2.33). iNOS staining co-localized with GnRH and oxytocin staining. In conclusion: The aging-related iNOS increased expression in the hypothalamus of the male rat affects regions known to control the synthesis and release of GnRH (POA, ARC) and oxytocin (PVN, SON), and the factors regulating penile erection (POA, and PVN). These observations suggest that iNOS may play a role in the reduction in GnRH and oxytocin neuronal secretion resulting in reproductive dysfunctions such as lowered serum testosterone, hypospermatogenesis, and diminished copulatory function in the aging male animal.

Alterations in the GnRH–LH system in relation to gonadal stage and Aroclor 1254 exposure in Atlantic croaker

Exposure of Atlantic croaker (Micropogonias undulatus) to the polychlorinated biphenyl mixture (Aroclor 1254, PCB; 1 mg/kg body wt/day for 30 days) during the early-recrudescence phase of the gonadal cycle results in the impairment of LH secretion and gonadal growth. In order to determine whether impairment was due to disruption of the stimulatory GnRH neuroendocrine pathway, we compared various parameters of the GnRH-LH system in early recrudescing vs. spermiating (mature) fish. Seabream GnRH (GnRH) content in the preoptic anterior hypothalamic area (POAH) and pituitary, pituitary GnRH receptor concentrations, and basal and GnRH analog (GnRHa)-induced LH secretion were significantly higher in gonadally mature croaker compared to early-recrudescing fish. In a subsequent experiment, the effects of PCB on the same neuroendocrine indices were investigated during the gonadal recrudescence phase of croaker. PCB exposure during the period of testicular maturation prevented the natural increase in GnRH content in the POAH but not in the pituitary. This finding suggests that PCB may impair GnRH synthesis in the POAH. The number of pituitary GnRH receptors also remained significantly lower in the PCB-exposed group, which was likely due to an impairment of GnRH release. The GnRH content in the POAH, number of pituitary GnRH receptors, and LH secretion in the PCB-exposed group were comparable to those in early-recrudescing fish, suggesting an impairment of normal maturation of the GnRH-LH system during the gonadal recrudescence phase. This impairment may be due to a direct action of PCB on GnRH neurons and/or indirectly via interference with other neurotransmitter pathways that modulate GnRH function.

Effects of chronic stress on hypothalamic lnterleukin-1beta, interleukin-2, and gonadotrophin-releasing hormone gene expression in ovariectomized rats.

The influence of chronic stress on the expression of interleukin (IL)-1beta and IL-2 mRNAs in ovariectomized rat brains, and the physiological consequences of the expression of these cytokines on hypothalamic-pituitary-gonadal (HPG) activity were investigated. Using polymerase chain reaction (PCR)-assisted semiquantitative analysis, we demonstrated alterated expression of IL-1beta and IL-2 mRNA during repeated cold stress; the expression of both IL-beta and IL-2 mRNA increased in the medial preoptic area and ventromedial hypothalamus, and decreased in the lateral hypothalamic area. In the arcuate nucleus/median eminence, IL-2 mRNA expression was dramatically decreased, in contrast to the increase in IL-1beta mRNA expression. Concomitant analysis of GnRH mRNA expression indicated significant suppression of GnRH synthesis in the chronic phase, and a strong negative correlation with cytokine expression in the medial preoptic area. Similar results were obtained in intact females exposed to this stress. These results, together with previous pharmacological studies, suggest that chronic stress may induce reproductive dysfunction through the effects of stress-induced expression of endogenous cytokines.

Estrogen directly respresses gonadotropin-releasing hormone (GnRH) gene expression in estrogen receptor-alpha (ERalpha)- and ERbeta-expressing GT1-7 GnRH neurons.

Estrogen has wide-ranging and complex effects on the reproductive axis, which are often difficult to interpret from in vivo studies. Estrogen negatively regulates tonic GnRH synthesis and also plays a pivotal role in the positive regulation of GnRH necessary for the preovulatory surge. To dissect the mechanisms by which these divergent effects occur, we attempted to observe the direct action of estrogen on the regulation of GnRH messenger RNA (mRNA) levels using the well characterized, GnRH-secreting, hypothalamic cell line, GT1-7. Using RT-PCR, we first investigated estrogen receptor transcript expression in GT1-7 neurons. We found that the GT1-7 cells express both estrogen receptor-alpha (ERalpha) and the recently described ERbeta mRNAs. We also detected the presence of both receptor subtypes in the GT1-7 neurons by Western blot analysis using specific ER antibodies. By Northern blot analysis of total GT1-7 RNA, we found that 17beta-estradiol (1 nM) down-regulates GnRH mRNA levels to approximately 55% of basal levels over a 48-h time course. This effect appears to occur specifically through an ER-mediated mechanism, as ICI 182,780, a complete ER antagonist, blocks the repression of GnRH mRNA levels by estradiol. The recently reported ERalpha-specific agonist/ERbeta-specific antagonist 2,2-bis-(p-hydroxyphenyl-1,1,1-trichloroethane (HPTE), a methoxychlor metabolite, also down-regulated GnRH gene expression. The repression of GnRH mRNA levels appears to occur at the transcriptional level, as simian virus 40 T antigen mRNA expression, which is under the control of 2.3 kb of the rat GnRH 5'-regulatory region, mimics the down-regulation of GnRH after treatment with estradiol. As the rat GnRH regulatory region in GT1-7 neurons does not appear to harbor a classic estrogen response element, the mechanism involved in the repression of GnRH has yet to be determined. These results suggest that estradiol directly regulates GnRH gene expression at the level of the GnRH neuron and may exert its neuroendocrine control through direct interaction with specific receptors expressed in these cells.

Activin A Regulation of Gonadotropin-Releasing Hormone Synthesis and Release in vitro

Activin is essential for the regulation of normal mammalian reproductive function at both the pituitary and gonadal levels. However, its central actions in the control of the hypothalamic-pituitary-gonadal axis remain largely unexplored. The present study aims to determine whether activin could regulate the reproductive axis at the level of the hypothalamus, through control of the GnRH neuroendocrine system. Using the GnRH-secreting GT1-7 neuronal cell line as a model system, we demonstrate expression of mRNAs encoding activin receptor types I, IB, and II. We examined the effects of activin A on GnRH protein secretion and mRNA levels in GT1-7 cells. Treatment with rh-activin A regulated both GnRH protein secretion and GnRH mRNA expression in the GT1-7 cells in a time-dependent fashion. Using transient transfection assays, we explored a potential transcriptional basis for these changes. Activin A increased reporter gene activity driven by minimal GnRH enhancer and promoter elements, suggesting that activin may regulate GnRH gene expression at the level of transcription. Lastly, activin A treatment of male rat hypothalami, in vitro, increased GnRH protein secretion. Collectively, molecular and physiological evidence support the presence of an activin system which might act at a hypothalamic site to regulate mammalian reproduction via activation of GnRH synthesis and release.

Promoter Transgenics Reveal Multiple Gonadotropin-Releasing Hormone-I-Expressing Cell Populations of Different Embryological Origin in Mouse Brain

Gonadotropin-releasing hormone-I (GnRH-I) is thought to be expressed by a single, highly spatially restricted group of neurons, which originate in the olfactory placode and migrate through the nose into the medial septum and hypothalamus from where they control fertility. Transgenic mice bearing a 13.5 kb GnRH-I-lacZ reporter construct were derived and found to express high levels of beta-galactosidase mRNA and protein within the septohypothalamic GnRH neurons in a correct temporal and spatial manner. Unexpectedly, low levels of beta-galactosidase were also present in three further populations of cells within the lateral septum, bed nucleus of the stria terminalis, and tectum. Analysis of wild-type mice with three different GnRH-I antibodies revealed distinct and transient patterns of GnRH-I peptide expression during development in all three of these populations revealed by transgenics. The synthesis of GnRH by cells of the lateral septum was the most persistent and remained until the third postnatal week. Embryonic "small eye" Pax-6 null mice, which fail to develop an olfactory placode, were also examined and shown to have equivalent populations of GnRH-I-immunoreactive cells in the lateral septum, tectum, and bed nucleus of the stria terminalis but none of the migrating cells that form the septohypothalamic GnRH population. These results prove that so-called "ectopic" expression in promoter transgenic lines can reflect authentic developmental patterns of gene expression. They further provide the first demonstration in mammalian brain that multiple neuronal populations of different embryological origin express GnRH-I peptide during embryonic and postnatal development.

An Analysis of Physiological Mechanisms Underlying the Antigonadotropic Action of Intracranial Prolactin in Ring Doves

Intracerebroventricular (ICV) injections of prolactin (PRL) exert potent antigonadal and antigonadotropic effects in ring doves (Streptopeliarisoria) at doses that are insufficient to stimulate prolactin-dependent crop growth. To explore the physiological basis of these effects, we tested the ability of ICV-injected PRL to influence pituitary responsiveness to chicken gonadotropin-releasing hormone-I (cGnRH-I) and to alter GnRH content and concentration in the preoptic area (POA) and median eminence (ME). cGnRH-I-induced changes in plasma LH were monitored by radioimmunoassay (RIA) in photostimulated male doves after they received five daily ICV injections of ovine PRL (1 μg/2 μl) or saline vehicle. Although PRL treatment reduced basal plasma LH levels and testes weight, it did not reduce the amount or alter the pattern of LH released in response to a bolus injection of cGnRH-I. This suggests that ICV PRL does not suppress LH by reducing pituitary responsiveness to GnRH. In two subsequent studies, GnRH content (ng/region) and concentration (pg/μg protein) in the POA and ME were measured in male doves by RIA and by competitive enzyme immunoassay after 5 days of ICV PRL or vehicle treatment. Although ICV PRL reduced plasma LH levels in both studies, no significant PRL-induced alterations in GnRH content or concentration were apparent. In a final study, PRL-treated female doves had lower plasma LH levels than vehicle-treated control females at 12 and 24 h after a single ICV injection. GnRH content of the POA was also lower in PRL-treated females than in controls at 24 h. However, the two treatment groups did not differ in POA or ME GnRH content at earlier postinjection sampling intervals. Analysis of GnRH concentration data revealed no treatment group differences in either region at any sampling interval (1, 6, 12, or 24 h post-PRL injection). Collectively, these results are consistent with the idea that ICV-injected PRL acts at the level of the CNS to inhibit the reproductive system. However, the nature of the alterations involved remains to be clarified. Plausible hypotheses are (1) that ICV PRL suppresses the gonadal axis by influencing the activity of GnRH neurons at brain sites other than the POA or ME or (2) that PRL alters the synthesis, storage, degradation, and/or release of GnRH in the POA or ME, but the dynamic changes involved are not reflected in integrated, steady-state measures such as peptide content or concentration in tissue.

Regional expression of mRNA encoding a second form of gonadotropin-releasing hormone in the macaque brain.

In mammals, reproduction is thought to be controlled by a single neuropeptide, gonadotropin-releasing hormone (GnRH-I), which regulates the synthesis and secretion of gonadotropins from the pituitary gland. However, another form of this decapeptide (GnRH-II), of unknown function, also exists in the brain of many vertebrate species, including humans; it is encoded by a different gene and its amino acid sequence is 70% identical to that of GnRH-I. Here we report the cloning of a GnRH-II cDNA from the rhesus macaque (Macaca mulatta), and show for the first time by in situ hybridization that GnRH-II mRNA is expressed in the primate midbrain, hippocampus and discrete nuclei of the hypothalamus, including the supraoptic, paraventricular, suprachiasmatic and arcuate. Because the regional distribution pattern of cells containing GnRH-II mRNA is largely dissimilar to that of cells containing GnRH-I mRNA, it is likely that these two cell populations receive distinct neuroendocrine inputs and thus regulate GnRH synthesis and release differently.

Changes in gonadotrophin-releasing hormone (GnRH-I) in the pre-optic area and median eminence of starlings (Sturnus vulgaris) during the recovery of photosensitivity and during photostimulation.

Changes in GnRH-I in the pre-optic (POA) and medio-basal (MBH) areas of the hypothalamus and in pituitary and plasma LH were measured in starlings (Sturnus vulgaris) during the recovery of photosensitivity under short days, and following photostimulation at various times during the recovery of photosensitivity. During exposure to short days there was a significant increase in GnRH-I in the POA, with the first detectable increase after only 10 days. There was no increase in GnRH-I in the MBH or in pituitary or plasma LH. In birds photostimulated after 10 short days, there was an increase in GnRH-I in the POA, but this was no greater than that in birds remaining under short days. There was no increase in GnRH-I in the MBH or in plasma LH. Photostimulation after 20 short days caused an immediate increase in GnRH-I in the POA, delayed increase in GnRH-I in the MBH, but no increase in plasma LH. Photostimulation after 30 short days caused an immediate increase in GNRH-I in the POA and the MBH and in plasma LH. The results show that the recovery of photosensitivity is gradual; the first measurable change occurs in the POA, consistent with photosensitivity being due to renewed GnRH-I synthesis. The effects of photostimulation increase, both in magnitude and in terms of how far 'downstream' of the POA changes are apparent, as photosensitivity is gradually restored. The results support the hypothesis that daylength has a dual role, controlling both synthesis and secretion of GnRH.

Gonadotropin-releasing hormone immunoreactivity in the nasal epithelia of adults with Kallmann's syndrome and isolated hypogonadotropic hypogonadism and in the early midtrimester human fetus.

GnRH-secreting neurons are known to originate in the epithelium of the medial olfactory placode, whence they migrate along the axons of the terminal nerve via the forebrain and into the hypothalamus. Synaptic contact between the developing olfactory bulbs and fascicles of the vomeronasal, terminal, and olfactory nerves does not occur in Kallmann's syndrome. Consequently, there is migration arrest of GnRH cells and partial or complete failure of formation of the olfactory bulbs, resulting in severe olfactory deficit and hypogonadotropic hypogonadism. In the present study, using an immunofluorescent, double immunostaining technique and confocal laser scanning microscopy, we observed GnRH-immunoreactive neurons in the hypothalamus of a 14-week-old human fetus. However, migration of GnRH neurons was not complete, and indeed, such cells were seen to be migrating along terminal nerve fascicles beneath the cribriform plate in a 16-week-old fetus. The same immunofluorescent technique demonstrated the presence of GnRH cells in biopsies of nasal mucosa obtained from three adults with Kallmann's syndrome, one normosmic subject with hypogonadotropic hypogonadism, and a eugonadal male cadaver. These findings are consistent with two different interpretations: the nasal GnRH neurons may be vestigial, representing cells that failed to migrate during embryogenesis; alternatively, they may have been generated de novo later in life, a possibility consistent with the recognized plasticity of human postnatal olfactory neuroepithelium. They also reveal that subjects with the normosmic (i.e. non-Kallmann's) form of GnRH deficiency are able to synthesize immunologically recognizable GnRH, implying that failure of GnRH synthesis is not responsible for this type of hypogonadotropic hypogonadism.

Stress and nutritional influences on GnRH and somatostatin neuronal systems in the ewe.

The effect of stressful stimulation and protein malnutrition on the gonadotrophic and somatotrophic axis of sheep is discussed with special references to the relationship between these stimuli and the GnRH and somatostatin neuronal systems in the hypothalamus. Generally, long-term stimulation and chronic underfeeding reduce gonadal functions in the sheep. There is evidence for the GnRH-dependent pathway for the mechanism of these phenomena in female sheep. GnRH neurons respond to long-term stress in diminishing of neuropeptide release from the nerve terminals due to the depression of its axonal transport. Chronic restriction of dietary proteins in lambs reduces the plasma LH concentrations but does not impair the development of GnRH neurons nor the synthesis and processing of GnRH. It is suggested that malnutrition delays the first ovulation probably due to the neural mechanism responsible for the preovulatory GnRH/LH output. Stress has rather unclear effect on growth hormone (GH) secretion in the sheep. Prolonged, but not short stressful stimulation provokes the rapid release of somatostatin, which is sustained during long-term stimulation. These results suggest that effect of stress on somatotrophic axis depends on the period of stressful stimulation. Chronic malnutrition enhances secretion of GH by an increase in amplitude of GH pulses and reduces the secretory activity of somatostatin neurons. It is postulated that nutrients can influence GH secretion in the sheep by mechanism dependent on the hypothalamic somatostatin.

Human chorionic gonadotropin down-regulates the expression of gonadotropin-releasing hormone receptor gene in GT1-7 neurons.

Previous studies have shown that human CG (hCG) can down-regulate the expression of GnRH gene in GT1-7 neurons and that these neurons contain GnRH receptors and a self-stimulatory mechanism in the synthesis and release of GnRH. These findings have led us to hypothesize that hCG may down-regulate GnRH receptors to disrupt the self-stimulatory mechanism. To test this hypothesis, we cultured GT1-7 neurons in the presence or absence of an optimal concentration of highly purified hCG (100 ng/ml) and then measured steady-state levels of GnRH receptor messenger RNA (mRNA) transcripts and protein. Northern blotting demonstrated that GT1-7 neurons contain a major 5.5-kb and minor 2.4-kb and 1.6-kb GnRH receptor mRNA transcripts. Ligand blotting showed that GT1-7 neurons also contain 53-kDa and 43-kDa GnRH receptor proteins. Culturing with hCG resulted in a significant decrease in steady-state levels of GnRH receptor mRNA transcripts by 12 h and GnRH receptor proteins by 6 and 12 h, followed by a return to the controls by 24 h. The treatment with hCG had no obvious effect on the transcription rate of GnRH receptor gene. The hCG treatment, however, significantly decreased the half-life of GnRH receptor mRNA transcripts from 27 h to 16 h. In summary, we conclude that treatment with hCG can down-regulate the expression of GnRH receptor gene by decreasing the stability of transcripts in GT1-7 neurons. By down-regulating GnRH receptors, hCG may disrupt the self-stimulatory mechanism in GnRH synthesis.