Gonadotropin-releasing hormone-I Research Articles

Effects of inorganic and organic manganese supplementation on gonadotropin-releasing hormone-I and follicle-stimulating hormone expression and reproductive performance of broiler breeder hens

Manganese is an essential microelement. Manganese deficiency affects reproduction performance and reproductive hormones in layers. However, little is known about its effects and the possible mechanism in regulating reproduction in broiler breeder hens. In the current study, broiler breeder hens at peak production were fed with diets supplemented with 0, 120, or 240 mg of Mn/kg as MnSO4 or Mn proteinate for 13 wk. Manganese supplementation did not alter egg laying rate, egg weight, fertility, hatchability, or hatchling weight over a 13-wk trial period. However, 240 mg of Mn/kg supplementation significantly increased serum Mn (P < 0.05). Manganese supplements increased the eggshell breaking strength (P < 0.05) without affecting the eggshell thickness. There was no difference in serum cholesterol and estradiol. Expression of follicle-stimulating hormone) and gonadotropin-releasing hormone-I (GnRH-I) genes was significantly elevated by 240 mg of Mn/kg (P < 0.05). Furthermore, inorganic Mn supplementation doubled GnRH-I expression compared with supplementation with the organic form (P < 0.05), although serum Mn was comparable between these 2 supplements. No obvious difference was shown in gene expression of luteinizing hormone, prolactin, GnRH-I receptor, inducible NO synthase, and dopamine receptor D1 in the pituitary as well as tyrosine hydroxylase and inducible NO synthase in the hypothalamus. This suggests that dietary Mn supplementation could improve eggshell quality in the long term. The central mechanism of nontoxic high doses of Mn suggested the transcriptional activation of GnRH-I and follicle-stimulating hormone genes. The central effect of inorganic Mn activating GnRH-I genes compared with the reduced effect by organic Mn could possibly be due to a decreased capacity of the latter passing through the blood-brain barrier.

Immunization against recombinant GnRH-I alters ultrastructure of gonadotropin cell in an experimental boar model

BackgroundGonadotropin cell is the main responsible for the secretion of follicle stimulating hormone (FSH) and luteinizing hormone (LH), and immunocastration reduces the concentrations of serum FSH and LH. A few studies have reported the histological structure of gonadotropin cells obtained from immunocastration animals at the light microscopy level. However, the ultrastructure of gonadotropin cells remains largely unexplored. The aim of this study was to evaluate and to compare ultrastructure of gonadotropin cell in gonadally intact boars and immunologically castrated male animals.FindingsIn this study, serum and adenohypophysis tissue were collected from nine gonadally intact boars and nine male pigs treated with recombinant gonadotropin releasing hormone I (GnRH-I). Anti-GnRH-I antibodies in serum and the ultrastructure of gonadotropin cell in adenohypophysis were determined by enzymelinked immunosorbent assay and electron microscopy, respectively. The results demonstrated that active immunization against recombinant GnRH-I increased serum GnRH-I antibody levels (P<0.05). Ultramicroscopic analysis of gonadotropin cell revealed a decrease (P<0.05) in the number and size of the large granules and small granules in the recombinant GnRH-I immunized animals.ConclusionsWe conclude that immunization against recombinant GnRH-I induces severe atrophy of granules in gonadotropin cell of boars, possibly reflecting GnRH-I regulation of gonadotropin cell.

Gonadotropin releasing hormone and brooding behavior in the native Thai hen

Changes in the number of hypothalamic gonadotropin releasing hormone-I (GnRH-I) neurons within the Nucleus commissurae pallii (nCPa) were associated with the reproductive cycle of native Thai chickens. In order to further understand the association of GnRH-I in the regulation of brooding behavior in this bird, the native Thai chickens were divided into two groups; chick-rearing (R) and non-chick-rearing (NR) hens. Numbers of visible of GnRH-I-immunoreactive (GnRH-I-ir) neurons in the hypothalamus of R and NR hens were compared utilizing immunohistochemistry. Numbers of visible GnRH-I-ir neurons within the Nucleus anterior medialis hypothalami, Nucleus suprachaiasmaticus, pars medialis, Nucleus septalis lateralis, Nucleus paraventricularis magnocellularis, and Regio lateralis hypothalami areas were observed in both groups, but no differences were seen between R and NR hens. The number of visible GnRH-I neurons in the nCPa was higher (P<0.05) in the NR than in R hens, and increased in NR hens by day 14 after chick removal. These findings suggest, for the first time, an association of the GnRH system with brooding behavior in continuously breeding birds. Furthermore, the expression of brooding behavior of native Thai chickens might be regulated, in part, by GnRH-I neurons in the nCPa.

Photoreceptive oscillators within neurons of the premammillary nucleus (PMM) and seasonal reproduction in temperate zone birds

The pathway for light transmission regulating the reproductive neuroendocrine system in temperate zone birds remains elusive. Based on the evidence provided from our studies with female turkeys, it is suggested that the circadian clock regulating reproductive seasonality is located in putatively photosensitive dopamine–melatonin (DA–MEL) neurons residing in the premammillary nucleus (PMM) of the caudal hypothalamus. Melanopsin is expressed by these neurons; a known photopigment which mediates light information pertaining to the entrainment of the clock. Exposure to a gonad stimulatory photoperiod enhances the activity of the DAergic system within DA–MEL neurons. DAergic activity encoding the light information is transmitted to the pars tuberalis, where thyroid-stimulating hormone, beta (TSHβ) cells reside, and induces the release of TSH. TSH stimulates tanycytes lining the base of the third ventricle and activates type 2 deiodinase in the ependymal which enhances triiodothyronine (T3) synthesis. T3 facilitates the release of gonadotropin-releasing hormone-I which stimulates luteinizing hormone/follicle stimulating hormone release and gonad recrudescence. These data taken together with the findings that clock genes are rhythmically expressed in the PMM where DA–MEL neurons are localized imply that endogenous oscillators containing photoreceptors within DA–MEL neurons are important in regulating the DA and MEL rhythms that drive the circadian cycle controlling seasonal reproduction.

Effects of repeated injection of kisspeptin-10 on the initiation of egg-laying in juvenile quail

The kisspeptin (Kp) signaling pathway plays an essential role in the onset of reproduction in mammals. To investigate the effects of Kp on the initiation of egg laying in birds, juvenile female quail were given daily intraperitoneal injections of 300μl saline (control, Con), or 10nmol (low dosage, L) or 100nmol (high dosage, H) kisspeptin-10 (Kp-10) dissolved in 300μl saline for 3 weeks. The ratio of egg laying of quail in the L and H groups was notably increased compared to that of the Con group (P<0.01), which paralleled earlier ovarian growth and increases in circulating estrogen (E2) concentrations. In the hypothalamus, gonadotropin-releasing hormone-I (GnRH-I) mRNA expression was markedly up-regulated, whereas the level of gonadotropin-inhibiting hormone mRNA was down-regulated by high-dose Kp-10 (P<0.05). In the pituitary gland, expression of GnRH receptor type II, but not type I mRNA was significantly up-regulated by high-dose Kp-10 administration (P<0.05). Moreover, compared with the Con group, follicle-stimulating hormone (FSH) gene expression in the pituitary was significantly decreased in the L and H groups (P<0.05), whereas luteinizing hormone (LH) mRNA expression was significantly increased in the H, but not the L group (P<0.05). These results indicate that repeated peripheral Kp-10 injections can advance the sexual maturation of female quail by regulating the activities of the hypothalamus–pituitary–gonadal axis.

Histone Deacetylases Regulate Gonadotropin-Releasing Hormone I Gene Expression via Modulating Otx2-Driven Transcriptional Activity

BackgroundPrecise coordination of the hypothalamic-pituitary-gonadal axis orchestrates the normal reproductive function. As a central regulator, the appropriate synthesis and secretion of gonadotropin-releasing hormone I (GnRH-I) from the hypothalamus is essential for the coordination. Recently, emerging evidence indicates that histone deacetylases (HDACs) play an important role in maintaining normal reproductive function. In this study, we identify the potential effects of HDACs on Gnrh1 gene transcription.Methodology/Principal FindingsInhibition of HDACs activities by trichostatin A (TSA) and valproic acid (VPA) promptly and dramatically repressed transcription of Gnrh1 gene in the mouse immortalized mature GnRH neuronal cells GT1–7. The suppression was connected with a specific region of Gnrh1 gene promoter, which contains two consensus Otx2 binding sites. Otx2 has been known to activate the basal and also enhancer-driven transcription of Gnrh1 gene. The transcriptional activity of Otx2 is negatively modulated by Grg4, a member of the Groucho-related-gene (Grg) family. In the present study, the expression of Otx2 was downregulated by TSA and VPA in GT1–7 cells, accompanied with the opposite changes of Grg4 expression. Chromatin immunoprecipitation and electrophoretic mobility shift assays demonstrated that the DNA-binding activity of Otx2 to Gnrh1 gene was suppressed by TSA and VPA. Overexpression of Otx2 partly abolished the TSA- and VPA-induced downregulation of Gnrh1 gene expression.Conclusions/SignificanceOur data indicate that HDAC inhibitors downregulate Gnrh1 gene expression via repressing Otx2-driven transcriptional activity. This study should provide an insight for our understanding on the effects of HDACs in the reproductive system and suggests that HDACs could be potential novel targets for the therapy of GnRH-related diseases.

The effect of active immunization against vasoactive intestinal peptide (VIP) and inhibin on reproductive performance of aging White Leghorn roosters

Decreasing fertility in aging domestic roosters is a well-known phenomenon. Aging is manifested by a decrease in plasma testosterone level, testis function, and spermatogenesis, resulting in a low level of fertility. The roles of vasoactive intestinal peptide (VIP) and testicular inhibin in this aging process are not clear. The effects of active immunization against VIP, inhibin, or the combination of both hormones on the reproduction of aging White Leghorn (WL) roosters were assayed. In experiment 1a, 60 White Leghorn roosters (67 wk of age) were divided into 4 groups (n = 15/group). The first group was actively immunized against VIP, the second against inhibin, the third against VIP and inhibin, and the fourth served as a control. Active immunization against VIP decreased semen quality parameters, plasma steroid levels, and gene expression of gonadotropin-releasing hormone-I (GnRH-I), follicle-stimulating hormone (FSH), luteinizing hormone (LH), LH receptor, VIP, and prolactin (Prl). Immunization against inhibin increased some of the semen quality parameters and FSH mRNA gene expression but decreased inhibin gene expression. In experiment 1b, at 94 wk of age, we took the actively immunized against VIP group and the control group and divided them into 2 subgroups (n = 7 or 8): the first group was injected with 1 mg of ovine Prl (oPrl) daily for 7 d, and the second group served as a control. Administration of oPrl to previously VIP-immunized birds significantly elevated semen quality parameters. We suggest that VIP, Prl, and inhibin have an important effect on the reproductive axis in aging roosters. Active immunization against VIP-depressed reproductive activity and Prl administration restored their reproduction, indicating that both VIP and Prl are essential for reproduction in aging roosters. Immunization against inhibin improved FSH mRNA gene expression, suggesting a negative role of inhibin on FSH secretion in aging roosters. Not all semen quality parameters increased significantly after immunization against inhibin, even though FSH mRNA gene expression increased, suggesting interference in testicular function in aging roosters.

The effect of active immunization against vasoactive intestinal peptide and inhibin on reproductive performance of young White Leghorn roosters

The neuropeptides vasoactive intestinal peptide (VIP) and gonadal inhibin have long been considered putative regulators of reproduction in hens. However, their role in young roosters remains unclear. We studied the effect of active immunization against VIP, inhibin, and a combination of both hormones on reproduction in young White Leghorn roosters. At 13 wk of age, White Leghorn roosters (n = 60) were split into 4 groups (n = 15). One group was actively immunized against VIP, the second against inhibin, the third against both VIP and inhibin, and the fourth, untreated, served as a control. Active immunization against VIP enhanced reproductive parameters as manifested by increased semen quality, plasma steroid levels, and mRNA gene expression of hypothalamic gonadotropin-releasing hormone-I, pituitary follicle-stimulating hormone, pituitary luteinizing hormone (LH), and decreased mRNA gene expression of hypothalamic VIP, pituitary prolactin, and testicular LH receptor. In contrast, immunization against inhibin decreased reproductive parameters such as semen quality, plasma steroid levels, mRNA gene expression of pituitary follicle-stimulating hormone and testicular inhibin. The combined treatment showed the greatest increase in semen quality parameters, plasma steroid levels, and mRNA gene expression of hypothalamic gonadotropin-releasing hormone-I, pituitary follicle-stimulating hormone, pituitary LH, and testicular LH receptor. Moreover, it significantly reduced mRNA gene expression of hypothalamic VIP and pituitary prolactin and mildly reduced that of testicular inhibin. These results suggest that VIP plays a negative role, at a young age, in reproduction of roosters that is similar to that in hens and that inhibin is as important in reproductive function in young roosters as in mammals.

Distribution and variation in gonadotropin releasing hormone-I (GnRH-I) immunoreactive neurons in the brain of the native Thai chicken during the reproductive cycle

Gonadotropin releasing hormone-I (GnRH-I) is known to regulate the avian reproductive system. We investigated the roles of GnRH-I in the regulation of the reproductive system of the native Thai chicken. The distribution of GnRH-I neurons and changes in GnRH-I-immunoreactive (-ir) neurons throughout the reproductive stages and between incubating and nest-deprived hens were analyzed utilizing immunohistochemical techniques. The results revealed that GnRH-I-ir neurons were distributed in a discrete region lying close to the third ventricle from the level of preoptic area through the anterior hypothalamus, with the greatest abundance found within the nucleus commissurae pallii (nCPa). The number of GnRH-I-ir neurons in the nCPa was highest in laying hens when compared with that in the other reproductive stages. Nest deprivation caused an increase in the number of GnRH-I-ir neurons in the nCPa of nest-deprived hens when compared with incubating hens. These results indicate that GnRH-I expression is correlated with the reproductive state in the native Thai chicken and may be, in part, regulated by it. This study also confirms a pivotal role of GnRH-I in controlling avian reproduction of this non-seasonal breeding, equatorial species.

Anandamide modulates the expression of GnRH-II and GnRHRs in frog, Rana esculenta, diencephalon

In the hypothalamus, endocannabinoids affect neuroendocrine activity by means of Gonadotropin-Releasing-Hormone-I (GnRH-I) inhibition. Since most vertebrates, human included, possess at least two GnRH molecular forms, the aim of this work was to investigate the effect of endocannabinoids on GnRH molecular forms other than GnRH-I and on GnRHRs.Thus, we cloned GnRH precursors as well as GnRH receptors (GnRHR-I, GnRHR-II, GnRHR-III) from the diencephalons of the anuran amphibian, Rana esculenta. GnRH-II expression was evaluated in pituitary, whole brain, spinal cord, hindbrain, midbrain and forebrain during the annual sexual cycle. Then, in post-reproductive period (May), GnRH-I, GnRH-II and GnRHRs expression was evaluated by quantitative real time (qPCR) after incubation of diencephalons with the endocannabinoid anandamide (AEA). AEA significantly decreased GnRH-I and GnRH-II expression, up regulated GnRHR-I and GnRHR-II mRNA and it had no effect upon GnRHR-III expression. These effects were counteracted by SR141716A (Rimonabant), a selective antagonist of type I cannabinoid receptor (CB1).In conclusion our results demonstrate a CB1 receptor dependent modulation of GnRH system expression rate (both ligands and receptors) in frog diencephalons. In particular, we show that AEA, besides GnRH-I, also acts on GnRH-II expression.

Potential roles for GNIH and GNRH-II in reproductive axis regulation of an opportunistically breeding songbird

The ability to breed at any time of year enables opportunistically breeding species to respond to good conditions whenever they occur. We investigate the neuroendocrine basis for this relatively unusual reproductive pattern in the avian world. One proposed mechanism for year-round breeding ability is tonic activation of gonadotropin-releasing hormone-I (GnRH-I) production that is flexibly modified by gonadotropin-inhibitory hormone (GnIH) production during unfavorable conditions. GnIH could inhibit GnRH secretion from the hypothalamus and/or inhibit GnRH action on the anterior pituitary gland. We studied neuroendocrine patterns in wild Australian zebra finches ( Taeniopygia guttata) sampled during a breeding period in Southern Australia, a non-breeding period in central Australia, and in juvenile males in the latter location. We asked whether patterns in immunoreactivity of three neuropeptides important for reproductive axis regulation, GnRH-I, GnRH-II and GnIH, during periods of breeding and non-breeding reflect this flexibility. We found that the numbers and sizes of immunoreactive (-ir) GnRH-I cells did not vary between breeding stages and ages. Contrary to our predictions, irGnIH cell number and size, as well as the synthesis of GnIH mRNA were similar in breeding and non-breeding conditions. However, breeding males had more and larger irGnRH-II cells in the midbrain compared to non-breeding males. Hence, while changes in irGnIH cells are not associated with fluctuations in gonadotropin secretion or gonad volume, the regulation of irGnRH-II cells might represent a previously-unidentified mechanism by which reproductive flexibility can be achieved; namely via behavioral neurotransmitter actions of GnRH-II rather than through the typical sensory-CNS integration-GnRH-I route.

Genetic effects of polymorphisms in candidate genes and the QTL region on chicken age at first egg

BackgroundThe age at first egg (AFE), an important indicator for sexual maturation in female chickens, is controlled by polygenes. Based on our knowledge of reproductive physiology, 6 genes including gonadotrophin releasing hormone-I (GnRH-I), neuropeptide Y (NPY), dopamine D2 receptor (DRD2), vasoactive intestinal polypeptide (VIP), VIP receptor-1 (VIPR-1), and prolactin (PRL), were selected as candidates for influencing AFE. Additionally, the region between ADL0201 and MCW0241 of chromosome Z was chosen as the candidate QTL region according to some QTL databases. The objective of the present study was to investigate the effects of mutations in candidate genes and the QTL region on chicken AFE.ResultsMarker-trait association analysis of 8 mutations in those 6 genes in a Chinese native population found a highly significant association (P < 0.01) between G840327C of the GnRH-I gene with AFE, and it remained significant even with Bonferroni correction. Based on the results of the 2-tailed χ2 test, mutations T32742394C, T32742468C, G32742603A, and C33379782T in the candidate QTL region of chromosome Z were selected for marker-trait association analysis. The haplotypes of T32742394C and T32742468C were significantly associated (P < 0.05) with AFE. Bioinformatics analysis indicated that T32742394C and T32742468C were located in the intron region of the SH3-domain GRB2-like 2 (SH3GL2) gene, which appeared to be associated in the endocytosis and development of the oocyte.ConclusionThis study found that G840327C of the GnRH-I gene and the haplotypes of T32742394C-T32742468C of the SH3GL2 gene were associated with the chicken AFE.

Polymorphisms associated with egg number at 300 days of age in chickens

We looked for variations that could be associated with chicken egg number at 300 days of age (EN300) in seven genes of the hypothalamic-pituitary-gonadal axis, including gonadotrophin-releasing hormone-I (GnRH-I), GnRH receptor (GnRHR), neuropeptide Y (NPY), dopamine D2 receptor (DRD2), vasoactive intestinal polypeptide (VIP), VIP receptor-1 (VIPR-1), prolactin (PRL), and the QTL region between 87 and 105 cM of the Z chromosome. Ten mutations in the seven genes were chosen to do marker-trait association analyses in a population comprising 1310 chickens, which were obtained from a company located in Guangdong Province of China. The C1704887T of VIPR-1 was found to have a highly significant association with EN300. The T5841629C of DRD2 and the C1715301T of VIPR-1 were significantly associated with EN300. A highly significant association was also found between the C1704887T-C1715301T haplotypes of VIPR-1 and EN300. H1H3 had the highest EN300. Four PCR-RFLP variations in the candidate QTL region were selected to investigate their genetic effects on EN300. The haplotypes of T32742468C-G32742603A in this region showed a highly significant association with EN300. Bioinformatics analyses showed that both T32742468C and G32742603A were located in intron 1 of the SH3-domain GRB2-like 2 (SH3GL2) gene. We conclude that five SNPs, including C1704887T and C1715301T of VIPR-1, T5841629C of DRD2, and T32742468C and G32742603A of SH3GL2, would be useful as markers for breeding to increase chicken EN300.

Seasonal changes in hypothalamic gonadotropin-releasing hormone-I immunoreactivity in relation with testicular volume in adult male free-living European starlings(Sturnus vulgaris)

Birds from the temperate and cold zones show annual sexual activity accompanied by gonadal changes and fluctuation in their brain gonadotropin-releasing hormone (GnRH) levels. However, most of the studies were done on captive birds where the constant environment can profoundly modify periodical changes. Therefore our aim was to reveal annual variations of hypothalamic and gonadal changes in male, free-living European starlings (Sturnus vulgaris) captured directly from their natural environment. We analyzed hypothalamic GnRH-I immunoreactivity and testes volume. Four key time points of the active reproductive cycle and the photorefractory phase were studied. GnRH-I immunoreactivity was analyzed in the preoptic area (POA) and the median eminence (ME). Photorefractory birds (August) with regressed gonads had the lowest level of GnRH-I immunoreactivity compared to other birds from the active reproductive phases. These results suggest that parallel with the gonadal volume GnRH-I undergoes seasonal changes in adult male free-living European starlings.

Identification of gonadotropin-inhibitory hormone in the zebra finch ( Taeniopygia guttata): Peptide isolation, cDNA cloning and brain distribution

Two novel RFamide peptides, kisspeptins and gonadotropin-inhibitory hormone (GnIH) are neuropeptides that appear critical in the regulation of the reproductive neuroendocrine axis. GnIH was first identified in avian brain, however, kisspeptins have not been identified in birds. To determine biochemically the presence of kisspeptins and GnIH in the zebra finch, a study was conducted to isolate these two peptides from zebra finch brain. Peptides were isolated by immunoaffinity purification and only one peptide was characterized by mass spectrometry. This peptide was confirmed to be a 12-amino acid sequence with RFamide at its C-terminus; its sequence is SIKPFSNLPLRFamide (zebra finch GnIH). By this approach, however, identification of kisspeptin from zebra finch brain was not achieved. Cloned zebra finch GnIH precursor cDNA encoded three peptides that possess characteristic LPXRFamide (X = L or Q) motifs at the C-termini. In situ hybridization and immunohistochemical analysis revealed the cellular localization of zebra finch GnIH mRNA and peptide in the paraventricular nucleus and the dorsomedial nucleus of the hypothalamus. Fluorescent immunohistochemistry with confocal microscopy indicated that GnIH-immunoreactive (ir) fibers are very close appositions with gonadotropin-releasing hormone-I (GnRH-I) cells. Furthermore GnIH-ir nerve fibers were widely distributed in the multiple brain regions including the septum, preoptic area, median eminence, optic tectum and median eminence. The prominent fibers were seen in the ventral tegmental area, midbrain central gray and dorsal motor nucleus of the vagus in the medulla. Thus, GnIH may participate in not only neuroendocrine functions but also regulation of motivation for social behavior and autonomic mechanisms.

The role of retinal and extra-retinal photostimulation in reproductive activity in broiler breeder hens

Photostimulation of retinal photoreceptors, which are sensitive to green light, appears to inhibit reproductive activity in birds, whereas photostimulation of extra-retinal photoreceptors, which are sensitive to red light, accelerates it. The objective of this study was to determine the effect of either retinal or extra-retinal photostimulation on reproductive activities of broiler breeder hens. At 23 wk of age, Cobb hens (N = 135) were divided into 9 rooms with individual cages (n = 15). At 24 wk of age, 3 rooms were photostimulated (14L:10D) with white light (Control, n = 45). Six rooms had 2 parallel lighting systems, red (660 nm) and green (560 nm), which were both on during 6 out of 14 h of the light period. Then, in 3 of these rooms, the green light was turned off and hens were exposed to a total of 14 h of red light (Red, n = 45), and in the other 3, the red light was turned off and green lighting continued for a total of 14 h (Green, n = 45). The Green group had reduced egg production; reduced plasma concentrations of ovarian steroids; reduced luteinizing hormone (LH)-β, vasoactive intestinal peptide (VIP), and prolactin mRNA expression; and greater retinal green opsin mRNA expression ( P ≤ 0.05). The Red group had greater egg production; greater gonadotropin-releasing hormone-I (GnRH-I) and red opsin gene expression in the hypothalamus; and lesser green opsin gene expression in the retina ( P ≤ 0.05). We suggest that selective photostimulation of extra-retinal photostimulation as opposed to retinal photostimulation is a key factor in the determination of successful reproduction of broiler breeder hens.

Effects of differential pulse frequencies of chicken gonadotrophin-releasing hormone-I (cGnRH-I) on laying hen gonadotrope responses in vitro

The aim of this work was to determine the effects of cGnRH I pulse frequencies on FSH and LH release and the changes in features and number of cultured laying hen FSH-cells and LH-cells in vitro. Primary adenohypophyseal cell cultures taken from laying hens were stimulated by four 5 min pulses using 1 or 10 nM cGnRH, administered with interpulses between pulses at 15, 30 or 60 min. Pulse frequencies and dose dependent effects were examined in six separate experiments including two controls. After the last interpulse time, the supernatants were collected and stored at −70° C until the performance of an indirect enzyme-linked immunosorbent assay (ELISA) using chicken LH and chicken FSH antisera at 1:1000 and 1:2000 dilutions, respectively. Supernatants were coated in duplicate on the inner surface of Immulon 2 plates and later blocked with the optimal solutions. They were incubated with each antiserum and subsequently with isotype-specific peroxidase-labeled anti-rabbit antibodies. Hydrogen peroxide/o-phenylenediamine was added as substrate/chromogen and the optical density (OD) was determined at 492 nm. The ABC immunocytochemical method was performed to characterize and re-count the gonadotropes employing anti-chicken FSH and anti-chicken LH as primary antibodies. The number of FSH-LH cells was obtained using stereological analysis and the data were statistically processed. The ODs obtained for each anti-hormone were compared with the control groups and with each other. Significant differences were found in number of aggregated-positive LH cells, which decreased with 1 nM cGnRH-I, 15 vs. 30 min pulses, increased with 30 vs. 60 min pulses, and also with 10 nM cGnRH-I, 30 vs. 60 min pulses. Aggregated positive FSH cells, however, did not show significant differences in percentage at any GnRH dose or pulse frequencies, but did show activity at low pulse frequencies of 15 and 30 min. The results suggest that LH cells varied in percentage in a dose dependent manner at higher pulse frequency (15 min) and were dose independent at low pulse frequency (60 min) and showed inactive features; while FSH cell numbers were unaffected showing features of activity at low pulse frequencies. High and moderate pulse frequencies of cGnRH-I (15-30 min) increased the FSH release in dose independent manner without changes in features or percentage of FSH cells. Low pulse frequency (60 min) of cGnRH-I increased LH release dose independently disminished LH cell percentage and showed changes in cells’ features. These results in avian cells showed differences in responses to GnRH pulse frequencies from those reported earlier in mammals.

Gonadotropin‐inhibitory hormone identification, cDNA cloning, and distribution in rhesus macaque brain

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide that modulates the reproductive physiology of birds and mammals by inhibiting gonadotropin secretion from the anterior pituitary gland. GnIH can also directly inhibit reproductive behaviors, possibly via action within the brain. Identification of the distribution of GnIH neurons and fibers may provide us with clues to how the brain controls reproductive activities of the animal. Here, we characterized the location and connectivity of GnIH neurons in the rhesus macaque (Macaca mulatta) brain. We determined the macaque GnIH precursor mRNA, and further identified a mature GnIH peptide (SGRNMEVSLVRQVLNLPQRF-NH(2)) by mass spectrometry combined with immunoaffinity purification. The majority of GnIH precursor mRNA-positive and GnIH-immunoreactive (GnIH-ir) cell bodies were localized in the intermediate periventricular nucleus (IPe) in the hypothalamus, as determined by in situ hybridization and immunocytochemistry, respectively. Abundant GnIH-ir fibers were observed in the nucleus of the stria terminalis in the telencephalon; habenular nucleus, paraventricular nucleus of the thalamus, preoptic area, paraventricular nucleus of the hypothalamus, IPe, arcuate nucleus of hypothalamus, median eminence and dorsal hypothalamic area in the diencephalon; medial region of the superior colliculus, central gray substance of the midbrain and dorsal raphe nucleus in the midbrain; and parabrachial nucleus in the pons. GnIH-ir fibers were observed in close proximity to gonadotropin-releasing hormone-I, dopamine, beta-endorphin, and gonadotropin-releasing hormone-II neurons in the preoptic area, IPe, arcuate nucleus of hypothalamus, and central gray substance of midbrain, respectively. GnIH neurons might thus regulate several neural systems in addition to pituitary gonadotropin release.

Anatomical localization of the effects of reproductive state, castration, and social milieu on cells immunoreactive for gonadotropin‐releasing hormone‐I in male European starlings (Sturnus vulgaris)

Gonadotropin-releasing hormone-I (GnRH-I) cells are localized primarily to the septopreoptic area (POA) and are responsible for regulating gonadotropin release from the anterior pituitary. Some songbird species exhibit dramatic seasonal variation in the number of detectable GnRH-I immunoreactive cells, with higher numbers being observed during the breeding season. Here we investigated the anatomical distribution of GnRH-I-immunoreactive cells in male starlings that varied in response to manipulations of reproductive state, social context, and gonadal condition. We housed photostimulated, intact and castrated male starlings with a female or alone. Additionally, a fifth treatment group consisted of photorefractory males (i.e., in a nonreproductive state) housed alone. All photostimulated males had significantly greater numbers of GnRH-I cells compared with photorefractory male starlings. There was a significant main effect of castration and social context. Castrated males had significantly greater numbers of GnRH-I cells compared with intact males, and males housed in male-female dyads also had greater numbers of GnRH-I cells. Furthermore, the significant main effects of castration and social context were the result of an increase in GnRH-I cell numbers specifically in the rostral and intermediate regions of the POA. These findings indicate that social context and hormonal milieu have profound effects on GnRH-I immunoreactivity in addition to the previously described effects of reproductive state. These data provide novel insight into the environmental regulation of the hypothalamopituitary axis and suggest that gonadal hormones and female presence independently regulate GnRH-I cells in specific regions of the POA in male starlings.

Is the gonadotropin releasing hormone system vulnerable to endocrine disruption in birds?

Endocrine disrupting chemicals (EDCs) from a variety of sources occur widely in the environment, but relationships between exposure to EDCs and long term effects on bird populations can be difficult to prove. Embryonic exposure to EDCs may be particularly detrimental, with potential long-term effects on reproduction and ultimately individual fitness. Because many EDCs may have subtle sublethal effects, it is necessary to establish sensitive end points as biomarkers of EDC exposure in birds. Because the effects of EDCs may be both short- and long-term, it is important to determine if embryonic exposure impacts sexual differentiation and development of the reproductive axis in hatchlings and if there are effects on reproductive function in adults. Our studies have focused on the effects of estrogen- and androgen-active EDCs on the hypothalamic gonadotropin releasing hormone–I (GnRH-I) system in an avian model of precocial species, the Japanese quail. Estrogen- or androgen-active EDCs were administered between 0 and embryonic day 4, and hypothalamic GnRH-I was measured in hatchlings and adults. Treatment with vinclozolin and PCB126 depressed the concentration of embryonic GnRH-I peptide while methoxyclor had an inconsistent stimulatory effect. Treatment with atrazine or trenbolone had no significant effects on hypothalamic GnRH-I in adults. Overall these observations support the view that the developing avian GnRH-I neural system may be vulnerable to EDCs with potential to alter lifelong reproductive function.