Gonadotropin-releasing Hormone mRNA Research Articles

Localization of Immunoreactive Gonadotropin-releasing Hormone and Relative Expression of Its mRNA in the Oviduct During Pregnancy in Rats

This study was designed to determine the cellular and ultrastructural distribution of the gonadotropin-releasing hormone (GnRH) and the relative expression of its mRNA in the oviduct of rats during different time points (days 7, 9, 16, and 20) of pregnancy. Immunofluorescent localization and confocal microscopic techniques were used to determine the cellular distribution of GnRH in the oviduct. Immunogold electron microscopy indicated its localization at the ultrastructural level, and real-time PCR was used to study the expression pattern of GnRH mRNA in the oviduct during pregnancy. In general, GnRH was localized within the epithelial cells lining the oviductal lumen at each selected time point. A strong correlation between the fluorescence intensity of GnRH-immunoreactive cells and the relative expression of GnRH mRNA was noted on days 7 and 16, followed by a plateau by day 20. At the ultrastructural level, uniform labeling of colloidal gold particles was observed in secretory vesicles and lamella of the luminal epithelium as well as the lumen of the oviduct. Collectively, these results demonstrate for the first time that the oviductal epithelium synthesizes and secretes the decapeptide GnRH during pregnancy in rats, which may have a possible role in postimplantation embryonic development and the maintenance of pregnancy.

Effect of acupuncture on pubertal development of rats and rabbits at different developmental stages

Physiological and endocrine studies on sexual development in animals and effects of acupuncture on sexual development are limited. Therefore, we investigated the effect of electro-acupuncture (EA) on the arcuate nucleus (Arc) and release of gonadotropin-releasing hormone (GnRH) in animals at different developmental stages. In Experiment 1, EA stimulation (30 Hz) was performed for 30 min per day in EA group of rabbits for 48 days, while the control group (mature rabbits) was not given EA. Arc discharges in those two groups were measured after the 48-day treatment. Arc discharge was also measured in the pre-pubertal group (as control) without EA treatment. Then, all three groups were treated with transient EA for 30 min and Arc discharges were determined again. In Experiment 2, EA (3 Hz) at the same acupoints or non-acupoints as that in the rabbits was performed for 20 min per day in different developmental group of Sprague-Dawley rats for 10 days. GnRH mRNA expression in the hypothalamus of rats was determined using RT-PCR and real-time PCR. The serum sexual hormone, sperm count, and body weight was measured. The results showed that the Arc discharge (P<0.01), testosterone (T) (P<0.01) and sperm count (P<0.01) in male rabbits were reduced by repeated EA. However, the body weight of rabbits was not changed after EA compared to the control in Experiment 1. In Experiment 2, GnRH mRNA expression in rats of the early pubertal group (EPG) and adult group (AG) were significantly depressed after repeated EA at acupoints (P<0.01). The sexual hormones were negatively influenced by repeated EA during puberty. Sperm count was reduced significantly after repeated EA at time of puberty (P<0.01). Repeated EA did not influence body weight of rats (P>0.01) and structures of the gonadial tissues during development. The results suggested that repeated EA is a good option that can be considered for regulating the function of the hypothalamus-pituitary-gonad (HPG) axis during puberty.

Orexin A Induces GnRH Gene Expression and Secretion from GT1-7 Hypothalamic GnRH Neurons

Orexin A, a recently discovered hypothalamic peptide, has been shown to have a stimulatory effect on release of gonadotropin-releasing hormone (GnRH) from rat hypothalamic explants in vitro. However, it is presently unclear whether in vivo this effect is mediated directly at the level of the GnRH neuron, or via multiple afferent neuronal connections. Therefore, in the present study, we investigated the direct action of orexin A on GnRH neurons using the immortalized GnRH-secreting GT1-7 hypothalamic cells. Orexin-1 receptor (OX1R) expression was detected in GT1-7 cells by RT-PCR and Western blot. Results showed that 0.1–1 nM orexin A, when administered in culture media for 4 h, can significantly stimulate GnRH mRNA expression in GT1-7 cells (p < 0.05). Administration of 1 µM OX1R antagonist, SB-334867, completely blocked the observed orexin A responses in these cells, indicating that orexin A stimulation of GnRH neurons is specifically through OX1R. Moreover, 0.1 nM orexin A stimulated GnRH release after 30–45 min. To examine possible signal transduction pathways involved in mediating these effects, a MEK inhibitor (UO-126), PKC inhibitor (calphostin C), and PKA inhibitor (H-89), were used, with each blocking orexin A-induced GnRH transcription and release from immortalized cells. Collectively, our results show that orexin A is capable of directly stimulating GnRH transcription and neuropeptide release from these immortalized hypothalamic neurons, and that the effects of orexin A appear to be mediated via the OX1R, coupled with activation of the PKC-, MAPK- and PKA-signaling pathways. It is suggested that the stimulatory effect of orexin A on GnRH transcription and release may also occur directly at the level of GnRH neurons in vivo.

Egr-1 binds the GnRH promoter to mediate the increase in gene expression by insulin

Insulin increases gonadotropin-releasing hormone (GnRH) gene expression in in vitro models of GnRH neurons. Early growth response-1 (Egr-1) is a transcription factor that mediates the effect of insulin on target genes. In the GN11 cell line – an immortalized GnRH-secreting neuronal cell line – insulin maximally increases Egr-1 mRNA after 30 min of treatment and Egr-1 protein and GnRH mRNA after 60 min of treatment. Egr-1 small interfering RNA blocks the insulin-induced increase in GnRH promoter activity, measured as luciferase expression. Chromatin immunoprecipitation using Egr-1 antibody precipitates DNA in a proximal region of the GnRH promoter but not DNA in a distal region. Mutagenesis of a putative Egr-1 binding site within the proximal region blocks the insulin-induced increase in GnRH promoter activity. Thus, Egr-1 binds the GnRH promoter at a site between −67 and −76 bp from the transcriptional start site to mediate the insulin-induced increase in GnRH gene transcription.

Ontogeny of gonadotropin‐releasing hormone (GnRH) neurons in hybrid striped bass Morone sp.: whole‐mount in situ hybridization analysis

The ontogeny of gonadotropin‐releasing hormone (GnRH) mRNA‐producing neurons was studied in developing hybrid striped bass (white bass Morone chrysops female × striped bass Morone saxatilis male), 1–55 days post‐fertilization (dpf), by whole‐mount in situ hybridization. Neurons that produce salmon (s) GnRH were first detected at 32 h post‐fertilization in the olfactory placodes. These neurons migrated posteriorly during development and reached their final position at the olfactory bulbs‐telencephalon boundary, possibly the terminal nerve ganglion (TNg) by 11 dpf. First signal of chicken (c) GnRH‐II neurons appeared in the midbrain 2 dpf and remained there throughout development. A signal of seabream (sb) GnRH mRNA was first detected at 21 dpf in the preoptic area (POA) and as a bilateral continuum along the ventral telencephalon at 32–55 dpf. The expression of all three forms of GnRH increased throughout development. These results suggest that cGnRH‐II neurons originate in the mid‐brain, and that sGnRH neurons originate in the olfactory placodes and migrate caudally to the TNg. Neurons that express sbGnRH seem to originate at the preoptic area and then to migrate anteriorly along the ventral telencephalon. An olfactory placodal origin of these neurons, however, cannot be ruled out.

A role for GnRH in early brain regionalization and eye development in zebrafish

Gonadotropin-releasing hormone (GnRH) is a highly conserved peptide that is expressed early in brain development in vertebrates. In zebrafish, we detected GnRH mRNA within 2 h post fertilization by RT-PCR. To determine if GnRH is involved in development, we used gene knockdown techniques to block translation of gnrh2 or gnrh3 mRNA after which the expression patterns for gene markers were examined at 24 h post fertilization with in situ hybridization. First, loss of either GnRH2 or GnRH3 affected regionalization of the brain as shown by a change in expression of fgf8 or pax2.1 genes in the midbrain–hindbrain boundary or diencephalon–midbrain boundary. Second, lack of GnRH2 and/or GnRH3 altered gene markers expressed in the formation of the eye cup ( pax2.1, pax6.1, mab21l2 and meis1.1) or eye stalk ( fgf8 and pax2.1). Third, knockdown of GnRH2 affected the size and shape of the midbrain and expression of gene markers therein. Results from assays with the TUNEL method and caspase-3 and -9 activity showed the brain and eye changes were unlikely to result from secondary apoptotic cell death before 24 h post fertilization. These experiments suggest that GnRH loss-of-function affects early brain and eye formation during development.

Effect of kisspeptin on GnRH mRNA expression in GT1-7 cells

During cholestatic liver disease, there is dysregulation in the balance between biliary growth and loss in bile duct–ligated (BDL) rats modulated by neuroendocrine peptides via autocrine/paracrine pathways. Gonadotropin-releasing hormone (GnRH) is a trophic peptide hormone that modulates reproductive function and proliferation in many cell types. We evaluated the autocrine role of GnRH in the regulation of cholangiocyte proliferation. The expression of GnRH receptors was assessed in a normal mouse cholangiocyte cell line (NMC), sham, and BDL rats. The effect of GnRH administration was evaluated in normal rats and in NMC. GnRH-induced biliary proliferation was evaluated by changes in intrahepatic bile duct mass and the expression of proliferation and function markers. The expression and secretion of GnRH in NMC and isolated cholangiocytes was assessed. GnRH receptor subtypes GnRHR1 and GnRHR2 were expressed in cholangiocytes. Treatment with GnRH increased intrahepatic bile duct mass as well as proliferation and function markers in cholangiocytes. Transient knockdown and pharmacologic inhibition of GnRHR1 in NMC decreased proliferation. BDL cholangiocytes had increased expression of GnRH compared with normal rats, accompanied by increased GnRH secretion. In vivo and in vitro knockdown of GnRH decreased intrahepatic bile duct mass/cholangiocyte proliferation and fibrosis. GnRH secreted by cholangiocytes promotes biliary proliferation via an autocrine pathway. Disruption of GnRH/GnRHR signaling may be important for the management of cholestatic liver diseases.

Effects of Chinese herbal medicine on modulating the course of puberty development in children with precocious puberty

To explore the therapeutic effects of Ziyin Xiehuo Recipe (ZYXHR) for nourishing yin and lowering fire and Yishen Tianjing Recipe (YSTJR) for nourishing kidney and replenishing essence on regulating the gonadotrophic and somatotrophic functions of hypothalamic-pituitary axis, and to reveal the mechanisms of ZYXHR and YSTJR in modulating the course of pubertal development of children with precocious puberty. The pubertal rats were fed with ZYXHR or YSTJR for 30 days, and the parameters of rats were monitored as the followings: The content of gonadotropin-releasing hormone (GnRH), the frequency and amplitude of GnRH impulse releasing, the releasing amounts of aminoacid neurotransmitters, and neuropeptide Y (NPY) and beta-endorphin (beta-END) in the gonadotrophic area of the hypothalamus were detected with neurobiological methods (push-pull perfusion, homogenate, incubation of brain slices, and immunohistochemical staining). The levels of gene and protein expressions of GnRH, growth hormone releasing hormone (GHRH) and somatostatin (SS) in hypothalamus, and follicular stimulating hormone (FSH), luteinizing hormone (LH) and growth hormone (GH) in adenohypophysis as well as insulin-like growth factor I (IGF-I) in metaphysis were determined with real time reverse transcription-polymerase chain reaction (RT-PCR). The ZYXHR could reduce the activity of GnRH neurons in hypothalamus through inhibiting the release of central exciting aminoacid neurotransmitters, whereas promoting the release of central inhibiting aminoacid neurotransmitters, NPY and beta-END in gonadotrophic area of hypothalamus. The expression levels of GnRH, FSH and LH mRNAs were down-regulated while the expression level of SS mRNA in hypothalamus was up-regulated in the ZYXHR-treated group. The GH mRNA in hypophysis and the IGF-I mRNA in metaphysis were both down-regulated by ZYXHR. The YSTJR could promote the activity of GnRH neurons in hypothalamus through inhibiting the release of NPY in gonadotrophic area of hypothalamus, up-regulating the expression levels of GnRH, FSH, LH and GH mRNAs in hypophysis, and IGF-I mRNA in metaphysis, while down-regulating the expression level of SS mRNA in hypothalamus. The ZYXHR and YSTJR could both regulate the gonadotrophic and somatotrophic functions of hypothalamic-pituitary axis through modulating the neuroendocrine regulation and the gene expressions of GnRH and SS in hypothalamus, GH, FSH and LH in hypophysis, and IGF-I in metaphysis. These may be the chief mechanisms of ZYXHR and YSTJR in modulating the course of pubertal development and ameliorating the skeletal development in children with precocious puberty.

Corticotrophin-releasing factor type 2 receptor-mediated suppression of gonadotrophin-releasing hormone mRNA expression in GT1-7 cells

Corticotrophin-releasing factor (CRF) released during stress has been implicated in the suppression of the hypothalamo–pituitary–gonadal (HPG) axis, especially the gonadotrophin-releasing hormone (GnRH) pulse generator, the central neural regulator of pituitary LH and FSH secretion, resulting in reproductive dysfunction. The gonadal steroid 17β-oestradiol (E2) has been shown to enhance CRF- and stress-induced suppression of pulsatile LH secretion. In the present study, we investigated the potential direct action of CRF on GnRH neurones by using GT1-7 cells, an established GnRH cell line. Furthermore, we investigated the modulatory influence of E2 on the effects of CRF and expression of CRF type 2 receptors (CRF-R2). Expression of CRF-R2 in the GT1-7 cells was detected by reverse transcription-polymerase chain reaction (RT-PCR). CRF produced a dose-dependent suppression of GnRH mRNA expression, an effect reversed by the selective CRF-R2 antagonist, astressin2-B (Ast2-B). E2 combined with CRF resulted in a greater suppression of GnRH expression compared with either treatment alone. E2 also increased CRF-R2 expression. These results demonstrate for the first time expression of CRF-R2 in the GT1-7 cells and suggest that CRF may directly regulate GnRH gene expression, an effect mediated, at least in part, by CRF-R2. They also raise the possibility that up-regulation of CRF-R2 may contribute to the sensitising influence of E2 on CRF- and stress-induced suppression of the GnRH pulse generator.

Luteinizing hormone, follicle stimulating hormone, and gonadotropin releasing hormone mRNA expression of Xenopus laevis in response to endocrine disrupting compounds affecting reproductive biology

Environmental pollutants can interfere with the endocrine system of a variety of animals and are suggested to contribute to the worldwide decline of amphibians. In this study, the effects of endocrine disrupting compounds (EDC) on the hypothalamus–pituitary–gonad axis, regulating reproduction, were investigated in Xenopus laevis by determining their potential impact on gene expression of gonadotropin releasing hormone (GnRH), luteinizing hormone β-subunit (LHβ) and follicle-stimulating hormone β-subunit (FSHβ) in brain and pituitary using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). One environmental sample and four model compounds, ethinylestradiol (EE2), tamoxifen (TAM), methyldihydrotestosterone (MDHT), and flutamide (FLU), corresponding to (anti)estrogenic and (anti)androgenic modes of action were used at 10 −8 M during a four weeks exposure of adults of both sexes. In general, males had a higher LHβ mRNA level compared to females, while the mRNA expression of FSHβ and GnRH did not differ between both sexes. EE2 and MDHT treatment decreased LHβ mRNA expression in the brain of male X. laevis, while only EE2 but not MDHT reduced LHβ mRNA in females indicating classical negative feed-back mechanisms on hypophyseal gonadotropin expression. TAM increased LHβ mRNA and FSHβ mRNA expression in female X. laevis while none of the other treatments showed an effect on FSHβ mRNA expression. GnRH expression was not changed by any treatment and exposure of X. laevis to Lambro river water had no significant effect on any of the genes examined. It is reported for the first time in amphibians that gonadotropin mRNA expression is differentially regulated by (anti)estrogenic and (anti)androgenic EDC and that gender-specific patterns of gene expression exist.

Evaluation of the true precocious puberty rats induced by neonatal administration of Danazol: therapeutic effects of nourishing "Yin"-Removing "Fire" Chinese herb mixture.

BackgroundNourishing "Yin"-Removing "Fire" Chinese Herb Mixture, a traditional herb-based formulation, has been successfully used for the management of idiopathic true precocious puberty (IPP) for more than thirty years. Precocious puberty rat model by neonatal administration of Danazol was used to investigate the effects of the herb mixture on the advanced sexual development of the rats, and the expression of hypothalamic gonadotropin-releasing hormone (GnRH), which is the important regulator for the hypothalamus-pituitary-gonadal axis, particularly at puberty.MethodsFemale Sprague-Dawley rats were divided into five groups: intact normal (N), IPP model (M), vehicle with no IPP (V), IPP model exposed to herb mixture (HM) and IPP model exposed to saline (S). Rats at 5 days of age were given a single subcutaneous injection of 300 microgram of Danazol dissolved in 25 microliter vehicle of propylene glycol-ethanol (1:1, v/v), to establish the precocious puberty model. From the day 15, rats in HM and S groups were continuously fed with either Nourishing "Yin"-Removing "Fire" Chinese Herb Mixture 2 ml or saline 2 ml, until 3 consecutive regular estrous cycles were established. The day of vaginal opening and the day of setup regular estrous cycle of the rats were observed. Blood concentration of estrogen was determined by radioimmunoassay. Immunohistochemistry and RT-PCR analysis were used to explore the expression of GnRH.ResultsThe day of vaginal opening and first estrous showed significant advancement in M compared with N and V (p < 0.05, respectively). The blood estrogen level increased significantly in M compared with those in other groups (about 28 days of age, at the time of vaginal opening in M rats) (p < 0.05, respectively). GnRH cells in rostral medial septum (MS), Broca diagonal band nucleus (DBB) and the medial preoptic area (MPOA), were calculated. The number in M was less than those in N and V (p < 0.05, respectively). The number was significantly higher in HM than that in M (p < 0.05). The GnRH mRNA expression increased significantly in M compared with that in N and V (p < 0.05).ConclusionThe true precocious puberty model by neonatal administration of Danazol in female rats showed augmented expression of hypothalamic GnRH; the Nourishing "Yin"-Removing "Fire" Chinese Herb Mixture down-regulated the increased GnRH expression, and significantly delayed the sexual development of the precocious puberty rat.

Effect of Calcitonin Gene‐Related Peptide on Gonadotrophin‐Releasing Hormone mRNA Expression in GT1‐7 Cells

Recent evidence has shown calcitonin gene-related peptide (CGRP) to be a key mediator of stress-induced suppression of the gonadotrophin-releasing hormone (GnRH) pulse generator, although little is known about the neural pathways involved. In the present study, we investigated the potential direct action of CGRP on GnRH neurones using GT1-7 cells, an established GnRH cell line. First, we detected expression of the CGRP receptor subunits, calcitonin receptor-like receptor and receptor activity-modifying protein-1 in the GT1-7 cells by reverse transcriptase-polymerase chain reaction. Second, we have shown that CGRP inhibits GnRH mRNA expression in the GT1-7 cells, which was effectively reversed by the CGRP receptor antagonist, CGRP8-37. These results suggest that CGRP down regulates expression of GnRH mRNA, via CGRP receptors in the GT1-7 cell, thus implying that a potential direct action of CGRP may mediate a suppressive effect on the GnRH neural network.

Differential gonadotropin releasing hormone (GnRH) expression, autoregulation and effects in two models of rat luteinized ovarian cells

GnRH has been suggested to participate in corpus luteum function. Here we studied the expression of GnRH mRNA and peptide in two models of rat luteinized tissues: ovarian cells from PMSG–hCG treated prepubertal rats (SPO) and from intrasplenic ovarian tumors (Luteoma). A GnRH autoregulatory effect was evaluated as well as its action on cell proliferation and apoptosis. GnRH mRNA was present in SPO, isolated corpora lutea from SPO and Luteoma from 1 week to 7 months of development. In vitro cultures of Luteoma cells expressed 2-fold higher GnRH mRNA and 10-fold higher GnRH peptide than SPO cells. Buserelin (GnRH analog) increased GnRH mRNA and peptide expression in SPO but not in Luteoma cells. While basal proliferation was very low in Luteoma cells, SPO cells showed a significant increase in cell number by both the thymidine and the MTS methods after 72 h in culture. Buserelin induced a decrease in cell number in both cell types to a similar degree. Although basal apoptosis levels were higher in SPO than in Luteoma cells, Buserelin-induced apoptosis was only detected in Luteoma cells after 48 h treatment. These results show that the two types of rat, luteinized tissues, Luteoma and SPO, markedly differed in some intrinsic properties and in their local GnRH systems. Luteoma cells proliferate very weakly, express and secrete high amounts of GnRH, do not show an autoregulatory effect and respond to the decapeptide with apoptosis stimulation. In contrast SPO cells proliferate significantly, secrete low levels of GnRH but possess a positive, autoregulatory mechanism and respond to GnRH stimulation with impairment of proliferation.

Differential Gonadotropin-Releasing Hormone (GnRH) and GnRH Receptor Messenger Ribonucleic Acid Expression Patterns in Different Tissues of the Female Rat across the Estrous Cycle

GnRH, the main regulator of reproduction, is produced in a variety of tissues outside of the hypothalamus, its main site of synthesis and release. We aimed to determine whether GnRH produced in the female rat pituitary and ovaries is involved in the processes leading to ovulation. We studied the expression patterns of GnRH and GnRH receptor (GnRH-R) in the same animals throughout the estrous cycle using real-time PCR. Hypothalamic levels of GnRH mRNA were highest at 1700 h on proestrus, preceding the preovulatory LH surge. No significant changes in the level of hypothalamic GnRH-R mRNA were detected, although fluctuations during the day of proestrus are evident. High pituitary GnRH mRNA was detected during the day of estrus, in the morning of diestrus 1, and at noon on proestrus. Pituitary GnRH-R displayed a similar pattern of expression, except on estrus, when its mRNA levels declined. Ovarian GnRH mRNA levels increased in the morning of diestrus 1 and early afternoon of proestrus. Here, too, GnRH-R displayed a somewhat similar pattern of expression to that of its ligand. To the best of our knowledge, this is the first demonstration of a GnRH expression pattern in the pituitary and ovary of any species. The different timings of the GnRH peaks in the three tissues imply differential tissue-specific regulation. We believe that the GnRH produced in the anterior pituitary and ovary could play a physiological role in the preparation of these organs for the midcycle gonadotropin surge and ovulation, respectively, possibly via local GnRH-gonadotropin axes.

The olfactory organ modulates gonadotropin-releasing hormone types and nest-building behavior in the tilapiaOreochromis niloticus

Direct olfactory inputs to any of the known gonadotropin-releasing hormone (GnRH) containing neurons have not been demonstrated. Therefore, the rationale of this study was to examine whether olfactory inputs might in some way interact with the GnRH system(s) to synchronize reproductive behaviors. In order to establish this, we used anosmic mature male tilapia to investigate changes in reproductive behaviors, gonadal morphology, and GnRH1, GnRH2, and GnRH3 cellular morphology and change in GnRH mRNA levels by real-time polymerase chain reaction. Bilateral removal of the olfactory rosettes followed by occlusion of the nasal cavity (ORX) inhibited nest-building behavior, but had no effect on aggressive and sexual behaviors or gonadal morphology. ORX failed to alter the morphological features of GnRH1, GnRH2, and GnRH3 (cell number, size, GnRH optical density), but significantly decreased copies of GnRH1 and GnRH2 mRNAs. GnRH immunoreactive fibers were not evident in the olfactory nerve and rosettes. DiI application to the olfactory nerve labeled inputs primarily to the glomerular layer of the olfactory bulbs and extrabulbar inputs to the forebrain but not to GnRH neurons. These results provide evidence that the olfactory rosette is crucial for modulating nest-building behavior through second-order olfactory pathways interacting with GnRH1 and GnRH2 neuronal systems.

Female pheromones stimulate release of luteinizing hormone and testosterone without altering GnRH mRNA in adult male Syrian hamsters ( Mesocricetus auratus)

In many species chemosensory stimuli function as important signals that influence reproductive status. Neurons synthesizing the peptide gonadotropin-releasing hormone (GnRH) are critical mediators of reproductive function via their regulation of the hypothalamic–pituitary–gonadal (HPG) axis, and they are thought to be responsive to chemosensory information. In the present study, we sought to elucidate the effects of female chemosensory stimuli on the HPG axis in sexually naive adult male Syrian hamsters. In Experiment 1, serial blood samples were collected from catheterized male hamsters following exposure to female pheromones in order to characterize the luteinizing hormone (LH) response to this chemosensory stimulus. In Experiment 2, brains and terminal blood samples were collected from animals 0, 60, and 120 min following pheromone exposure. GnRH mRNA was measured in brain tissue sections using in situ hybridization, and plasma concentrations of LH and testosterone were measured using radioimmunoassay. Data from Experiment 1 indicated that female pheromones elicited a rapid rise in plasma LH that peaked at 15 min and returned to baseline 45 min after exposure. In Experiment 2, testosterone was elevated in terminal blood samples obtained 60 min, but not 120 min, after exposure to pheromones. LH levels were unaffected at both of these time points. The chemosensory-induced increases in LH and testosterone release were not accompanied by subsequent changes in GnRH mRNA over the time course studied. These data suggest that while activation of the male HPG axis by female pheromones involves release of GnRH, it does not involve increases in GnRH mRNA 1–2 h after pheromonal stimulation as a mechanism for replenishment of released peptide.

Dietary daidzein influences laying performance of ducks ( Anas platyrhynchos) and early post-hatch growth of their hatchlings by modulating gene expression

Our previous studies demonstrated that dietary supplementation of daidzein improves egg production in duck breeders during late periods of the laying cycle. The present study was aimed to clarify whether the growth of ducklings hatched from eggs laid by daidzein-treated hens would be affected, and to elucidate the mechanisms underlying potential trans-generational effects, by determining changes of hormone levels and mRNA expression of relevant genes. Daidzein was added to the basal diet of 415-day-old duck breeders at the level of 5 mg/kg. During 9 weeks of daidzein treatment, laying rate increased by 7.70%, average egg mass tended to increase, whereas yolk/albumen ratio decreased significantly. These changes were accompanied by significantly elevated plasma T 4 and E 2 levels, enhanced gonadotropin releasing hormone (GnRH) mRNA, but diminished estrogen receptor (ER)-β mRNA expression in hypothalamus of daidzein-treated hens. Ducklings hatched from daidzein-treated eggs were significantly smaller at hatching, but they caught up with their control counterparts by 4 weeks of age. Serum levels of T 4, pituitary GH, hepatic GH receptor (GHR) and type-1 IGF receptor (IGF-1R) mRNA expression were all suppressed markedly in the daidzein-treated group at hatching, but this suppression proved to be temporary, as at 4 weeks of age, expression levels of all investigated genes were restored. However, it is noteworthy that at 4 weeks of age an obvious down-regulation of hypothalamic GnRH mRNA expression was detected in ducklings maternally exposed to daidzein. Our results provide evidence that maternal exposure to daidzein affects post-hatch growth in the duck with accompanying changes in the secretion of metabolic hormones and expression of growth-related genes. Although the negative effect of maternal daidzein on embryonic growth could be eliminated 4 weeks after hatching, the long-term effect of maternal daidzein on reproductive function is not to be ignored and awaits further investigation.

Changes in brain GnRH mRNA and pituitary GnRH peptide during testicular maturation in barfin flounder

The pleuronectid barfin flounder ( Verasper moseri) expresses three forms of gonadotropin-releasing hormone (GnRH) in the brain. To clarify the physiological roles of the respective forms during testicular maturation, changes in brain GnRH mRNA levels and pituitary GnRH peptide levels were examined by real-time quantitative PCR and time-resolved fluoroimmunoassay, respectively. Fish hatched in April 2000. The gonadosomatic index remained low until October 2001 and then rapidly increased in January 2002. Fish continued to grow from hatching through testicular maturation. Fish spermiated in March 2002. The amount of seabream GnRH (sbGnRH) mRNA per brain significantly increased in January 2002 and remained at high levels in March 2002. The amounts of salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II) mRNA per brain did not show significant changes during the experimental periods. Pituitary sbGnRH peptide content significantly increased in March 2002. Pituitary sGnRH peptide and cGnRH-II peptide contents were extremely low compared to sbGnRH peptide levels and showed no significant changes during the experiment. These results indicate that sbGnRH is involved in the testicular maturation of barfin flounder.

In vivo action of a new octadecaneuropeptide (ODN) antagonist on gonadotropin-releasing hormone gene expression in the male rat brain

It has been reported that several of the effects induced by octadecaneuropeptide (ODN) could be mediated by an activation of a metabotropic receptor. In order to investigate the role and mechanism of action of ODN in gonadotropin-releasing hormone (GnRH) neuron regulation, we studied the effects of the acute i.c.v. administration of ODN and of a new ODN antagonist to metabotropic receptor, cyclo 1–8[Dleu 5]OP, on GnRH mRNA expression as evaluated by in situ hybridization in castrated male rats. The administration of ODN produced a decrease in the hybridization signal while the administration of cyclo 1–8[Dleu 5]OP alone produced an 18% increase. When administrated concomitantly with ODN, the antagonist both inhibited the depressing effect of ODN and induced a 22% increase over the values detected in ODN-treated rats. The data suggest that the effect of ODN on GnRH mRNA expression might be mediated by interaction with metabotropic receptors.

Increased expression of forebrain GnRH mRNA and changes in testosterone negative feedback following pubertal maturation

Pubertal development is associated with increased activity of the gonadotropin releasing hormone (GnRH) neuronal system and rising gonadal steroid levels. The purpose of this study was to determine whether different circulating levels of testosterone affect GnRH mRNA and luteinizing hormone (LH) to the same degree prior to and following pubertal maturation. Pre- and post-pubertal male Syrian hamsters were gonadectomized and treated with timed-release testosterone pellets (0, 0.5, 1.5, or 2.5 mg) for one week. Following treatment, three separate brain tissue dissections containing the majority of GnRH cell bodies, tenia tecta and medial septum (TT/MS), diagonal band of Broca/organum vasculosum of the lamina terminalis (DBB/OVLT), and preoptic area (POA), were analyzed for GnRH mRNA levels by RNase protection assay and terminal plasma luteinizing hormone concentrations were determined by radioimmunoassay. Pre-pubertal animals were more sensitive to testosterone negative feedback on LH. Conversely, the ability of testosterone to reduce GnRH mRNA was much greater after pubertal development. Specifically, GnRH mRNA in the TT/MS was considerably higher in adults, and testosterone reduced GnRH mRNA in a dose-dependent manner only in adults. These data indicate that although testosterone is a powerful suppressor of LH release before puberty, it does not have appreciable control over GnRH mRNA until after puberty. Furthermore, the pubertal increase in GnRH mRNA appears to occur via steroid feedback-independent mechanisms in the male Syrian hamster.