Grégoy Y. Bédécarrats

Gonadotropin-inhibitory hormone (GnIH) and its control of central and peripheral reproductive function.

Identification of novel neurohormones that regulate the reproductive axis is essential for the progress of neuroendocrinology. The decapeptide gonadotropin-releasing hormone (GnRH) is the primary factor responsible for the hypothalamic control of gonadotropin secretion. Gonadal sex steroids and inhibin modulate gonadotropin secretion via feedback from the gonads, but a neuropeptide that directly inhibits gonadotropin secretion was unknown in vertebrates until 2000 when a hypothalamic dodecapeptide serving this function was discovered in quail. Because of its action on cultured pituitary in quail, it was named gonadotropin-inhibitory hormone (GnIH). GnIH acts on the pituitary and on GnRH neurons in the hypothalamus via a novel G protein-coupled receptor (GPR147). GPR74 may also be a possible candidate GnIH receptor. GnIH decreases gonadotropin synthesis and release, inhibiting gonadal development and maintenance. Melatonin stimulates the expression and release of GnIH via melatonin receptors expressed by GnIH neurons. GnIH actions and interactions with GnRH seem common not only to several avian species, but also to mammals. Thus, GnIH is considered to have an evolutionarily conserved role in controlling vertebrate reproduction, and GnIH homologs have also been identified in the hypothalamus of mammals. As in birds, mammalian GnIH homologs act to inhibit gonadotropin release in several species. More recent evidence in birds and mammals indicates that GnIH may operate at the level of the gonads as an autocrine/paracrine regulator of steroidogenesis and gametogenesis. Importantly, GnIH in birds and mammals appears to act at all levels of the hypothalamo-pituitary-gonadal (HPG) axis, and possibly over different time-frames (minutes-days). Thus, GnIH and its homologs appear to act as key neurohormones controlling vertebrate reproduction. The discovery of GnIH has enabled us to understand and manipulate vertebrate reproduction from an entirely new perspective.

Activation of the chicken gonadotropin-inhibitory hormone receptor reduces gonadotropin releasing hormone receptor signaling.

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic peptide from the RFamide peptide family that has been identified in multiple avian species. Although GnIH has clearly been shown to reduce LH release from the anterior pituitary gland, its mechanism of action remains to be determined. The overall objectives of this study were (1) to characterize the GnIH receptor (GnIH-R) signaling pathway, (2) to evaluate potential interactions with gonadotropin releasing hormone type III receptor (GnRH-R-III) signaling, and (3) to determine the molecular mechanisms by which GnIH and GnRH regulate pituitary gonadotrope function during a reproductive cycle in the chicken. Using real-time PCR, we showed that in the chicken pituitary gland, GnIH-R mRNA levels fluctuate in an opposite manner to GnRH-R-III, with higher and lower levels observed during inactive and active reproductive stages, respectively. We demonstrated that the chicken GnIH-R signals by inhibiting adenylyl cyclase cAMP production, most likely by coupling to G(alphai). We also showed that this inhibition is sufficient to significantly reduce GnRH-induced cAMP responsive element (CRE) activation in a dose-dependent manner, and that the ratio of GnRH/GnIH receptors is a significant factor. We propose that in avian species, sexual maturation is characterized by a change in GnIH/GnRH receptor ratio, resulting in a switch in pituitary sensitivity from inhibitory (involving GnIH) to stimulatory (involving GnRH). In turn, decreasing GnIH-R signaling, combined with increasing GnRH-R-III signaling, results in significant increases in CRE activation, possibly initiating gonadotropin synthesis.

Identification of a Novel Pituitary-Specific Chicken Gonadotropin-Releasing Hormone Receptor and Its Splice Variants

Abstract In all vertebrates, GnRH regulates gonadotropin secretion through binding to a specific receptor on the surface of pituitary gonadotropes. At least two forms of GnRH exist within a single species, and several corresponding GnRH receptors (GNRHRs) have been isolated with one form being pituitary specific. In chickens, only one type of widely expressed GNRHR has previously been identified. The objectives of this study were to isolate a chicken pituitary-specific GNRHR and to determine its expression pattern during a reproductive cycle. Using a combined strategy of PCR and rapid amplification of cDNA ends (RACE), a new GNRHR (chicken GNRHR2) and two splice variants were isolated in domestic fowl (Gallus gallus domesticus). Full-length GNRHR2 and one of its splice variant mRNAs were expressed exclusively in the pituitary, whereas mRNA of the other splice variant was expressed in most brain tissues examined. The deduced amino acid sequence of full-length chicken GNRHR2 reveals a seven transmembrane domain protein with 57%–65% homology to nonmammalian GNRHRs. Semiquantitative real-time PCR revealed that mRNA levels of full-length chicken GNRHR2 in the pituitary correlate with the reproductive status of birds, with maximum levels observed during the peak of lay and 4 wk postphotostimulation in females and males, respectively. Furthermore, GnRH stimulation of GH3 cells that were transiently transfected with cDNA that encodes chicken GNRHR2 resulted in a significant increase in inositol phosphate accumulation. In conclusion, we isolated a novel GNRHR and its splice variants in chickens, and spatial and temporal gene expression patterns suggest that this receptor plays an important role in the regulation of reproduction.

Gonadotropin-inhibitory hormone receptor signaling and its impact on reproduction in chickens.

In birds, as in other vertebrates, reproduction is controlled by the hypothalamo-pituitary-gonadal axis with each component secreting specific neuropeptides or hormones. Until recently, it was believed this axis is exclusively under the stimulatory control of hypothalamic gonadotropin-releasing hormone I (GnRH-I) which in turn, stimulates luteinizing hormone (LH) and follicle stimulating hormone (FSH) secretion from the pituitary gland. However, the discovery of a novel inhibitory hypothalamic peptide able to reduce LH secretion (gonadotropin-inhibitory hormone: GnIH) challenged this dogma. Furthermore, with the characterization of its specific receptor (GnIHR), progress has been made to clarify the physiological relevance of GnIH in birds. This short review discusses the recent advances in GnIHR signaling at the level of the pituitary gland and the gonads. GnIHR is a member of the G-protein coupled receptor (GPCR) family which couples to G(alphai) and, upon activation inhibits adenylyl cyclase (AC) activity, thus reducing intracellular cAMP levels. This implies that GnIH interferes with signaling of any GPCR coupled to G(alphas), including GnRH, LH and FSH receptors. In the chicken pituitary gland, the GnRHR-II/GnIHR ratio changes during sexual maturation in favor of GnRHR-II that appears to result in hypothalamic control of gonadotropin secretion shifting from inhibitory to stimulatory, with corresponding changes in GnRH-induced cAMP levels. Within the gonads, GnIH and its receptor may act in an autocrine/paracrine manner and may interfere with LH and FSH signaling to influence ovarian follicular maturation and recruitment, as well as spermatogenesis.

Red light is necessary to activate the reproductive axis in chickens independently of the retina of the eye

Photoperiod is essential in manipulating sexual maturity and reproductive performance in avian species. Light can be perceived by photoreceptors in the retina of the eye, pineal gland, and hypothalamus. However, the relative sensitivity and specificity of each organ to wavelength, and consequently the physiological effects, may differ. The purpose of this experiment was to test the impacts of light wavelengths on reproduction, growth, and stress in laying hens maintained in cages and to determine whether the retina of the eye is necessary. Individual cages in 3 optically isolated sections of a single room were equipped with LED strips providing either pure green, pure red or white light (red, green, and blue) set to 10 lx (hens levels). The involvement of the retina on mediating the effects of light wavelength was assessed by using a naturally blind line (Smoky Joe) of chickens. Red and white lights resulted in higher estradiol concentrations after photostimulation, indicating stronger ovarian activation, which translated into a significantly lower age at first egg when compared with the green light. Similarly, hens maintained under red and white lights had a longer and higher peak production and higher cumulative egg number than hens under green light. No significant difference in BW gain was observed until sexual maturation. However, from 23 wk of age onward, birds exposed to green light showed higher body growth, which may be the result of their lower egg production. Although corticosterone levels were higher at 20 wk of age in hens under red light, concentrations were below levels that can be considered indicative of stress. Because no significant differences were observed between blind and sighted birds maintained under red and white light, the retina of the eye did not participate in the activation of reproduction. In summary, red light was required to stimulate the reproductive axis whereas green light was ineffective, and the effects of stimulatory wavelengths do not appear to require a functional retina of the eye.

An updated model to describe the neuroendocrine control of reproduction in chickens.

Since its first identification in quail 15 years ago, gonadotropin inhibitory hormone (GnIH) has become a central regulator of reproduction in avian species. In this review, we have revisited our original model published in 2009 to incorporate recent experimental evidence suggesting that GnIH acts as a molecular switch during the integration of multiple external and internal cues that allow sexual maturation to proceed in chickens. Furthermore, we discuss the regulation of a dual inhibitory/stimulatory control of the hypothalamo-pituitary-gonadal axis involving the interaction between GnIH and gonadotropin releasing hormone (GnRH). Finally, beyond seasonality, we also propose that GnIH along with this dual control may be responsible for the circadian control of ovulation in chickens, allowing eggs to be laid in a synchronized manner.

Development, validation, and utilization of a novel antibody specific to the type III chicken gonadotropin-releasing hormone receptor.

Two gonadotropin-releasing hormone receptors (GnRH-Rs) have been characterized in chickens to date: cGnRH-R-I and cGnRH-R-III, with cGnRH-R-III being the predominant pituitary form. The purpose of the present study was to first validate a novel antibody for the specific detection of cGnRH-R-III and second, using this antibody, detect changes in cGnRH-R-III protein levels in the pituitary gland of male and female chickens during a reproductive cycle. The localization of cGnRH-R-III within the anterior pituitary gland was also determined. Western blotting of pituitary extracts and transiently transfected COS-7 cell lysates revealed that our antibody is highly specific to cGnRH-R-III protein. Similarly, when used in immunocytochemistry, this antibody specifically detects cells expressing cGnRH-R-III and not cGnRH-R-I. Western blot analyses of chicken pituitary gland homogenates show that cGnRH-R-III protein levels are significantly greater in sexually mature birds than in immature birds or birds at the end of a reproductive cycle (P < 0.0001). A similar pattern was observed for both males and females. Additionally, the antibody was able to detect cGnRH-R-III in cells along the periphery of the cephalic and caudal lobes of the anterior pituitary where the cells containing the gonadotropins are located. In summary, we successfully validated a novel antibody to cGnRH-R-III and showed levels of cGnRH-R-III protein in the pituitary fluctuate with respect to the reproductive status in both male and female chickens.

Prolactin and its Receptor in Galliformes

In recent years, the range of reported actions ofhormones has considerably expanded. The adenohy-pophyseal hormone, prolactin (PRL), however, is stillunrivalled in the extraordinary diversity of biologicalactions which have been attributed to it. The first roleattributed to PRL was the induction of milk secretionin rabbits (Stricker and Greuter, 1928) and in 1935, itwas shown that PRL was a causative factor in theinduction of incubation behaviour in ‘‘broody strains’’of actively laying chickens (Riddle et al., 1935). Sincethen, the roles of PRL have considerably expanded anda recent review categorizes over 300 separate actionsinto those which modulate: (1) water and electrolytebalance, (2) growth and development, (3) endocri-nology and metabolism, (4) brain and behaviour, (5)reproduction and (6) immunoregulation and protection(Bole-Feysot et al., 1998).One of the most studied roles of PRL in avesinvolves the incubation phase of broody behaviour.Broodiness occurs during the breeding season in manyavian species and consists of 2 phases: the incubationof eggs and the raising of young. Many studies haveindicated that hyperprolactinemia is associated withthe onset and maintenance of incubation behaviour inchickens and turkeys. The role(s) that PRL may effectduring this behaviour are not known but immunolo-gical evidence (suppression of PRL levels via active orpassive immunization against PRL or its majorreleasing factor, VIP (Sharp, 1997; Criso´stomo et al.,1997, 1998) clearly suggest that high levels of PRL area requisite aspect of this behaviour. Since the expres-sion of incubation behaviour is also associated withlarge changes in physiology including gonadal involu-tion, aphagia and adipsea it may be expected that PRLmay have multiple roles in mediating adaptions to thebehaviour. How does a single hormone cause so manydifferent responses? In this review, we will brieflyconsider functional aspects of the genes encoding PRLand its receptor, changes in glycosylation patterns ofPRL and genetic variation in the genes.

Inner retinal cell development is impaired in Smoky Joe chickens.

Many different components of the retina can be affected by inherited degenerative diseases causing blindness. Currently, 5 different mutant strains of chicken have already been studied as potential models for inherited retinal degeneration; however, the potential for the blind strain of White Leghorns, called Smoky Joe (SJ), as a model remains unknown. Ocular symptoms observed within homozygous SJ birds show the birds have varying levels of blindness at hatch and by 8 wk posthatch are completely blind, but details about the development of the blindness are unclear (Salter et al., 1997). The objective of this study was to characterize the retinal development of blind and sighted SJ chicks during embryogenesis, and to monitor the numbers of the retinal cells with cell-type-specific markers. Blind SJ chicks showed less retinal cells throughout embryogenesis compared with sighted SJ chicks (P < 0.0001). Based on the histological analysis, it was determined that amacrine cells within the inner nuclear layer were the most affected cell type, showing lower numbers in the blind SJ compared with the sighted; amacrine cell development was also delayed in the blind birds, beginning 2 d later than in sighted SJ birds. Photoreceptors were also scarcely detected within the blind SJ and potentially may be an additional target of developmental impairment. Further analysis on posthatch SJ will aid in determining degenerative characteristics of a fully developed retina and its cells.

Use of dietary thyroxine as an alternate molting procedure in spent turkey breeder hens

In the turkey industry, molting is traditionally achieved by reducing photoperiod and withdrawing feed and water for several days. Although it is the most effective method, this practice is discouraged in Canada and alternative strategies need to be established. Thyroid hormone levels naturally change during molt, and dietary thyroxine (T4) supplementation was previously shown to induce molt in chickens. This study aimed at evaluating the effectiveness of supplemental dietary T4 in inducing molt in spent turkey breeder hens. One hundred twenty 75-wk-old hens were randomly divided into 4 groups (5 floor pens/replicates, 5 hens each) with the control group kept under a 14-h photoperiod and fed a breeders diet throughout, whereas hens from the 3 other groups were supplemented with 40 ppm (45.76 mg/kg) T4 for 10 d. One treatment group was maintained under 14 h of light and fed a breeders diet, whereas the 2 others were subjected to a drop in photoperiod to 6 h during or after supplementation and then were fed a maintenance diet. Egg production, feed intake, BW, molt, and plasma levels of T4, prolactin, and luteinizing hormone were measured. All treated hens ceased laying by d 20; however, several individuals spontaneously returned to lay when left on 14 h of light, suggesting incomplete involution of the reproductive tract. Supplementation significantly reduced feed consumption and induced rapid BW loss. All hens returned to their initial weight by the end of the experiment. Most treated hens initiated molt by d 8 of supplementation and all completed molt by d 37. Plasma T4 in treated hens increased significantly by d 3 (P < 0.05) and remained significantly higher than in controls until d 9 (P < 0.01). Levels returned to initial values by d 35. Prolactin levels did not appear to be influenced by T4 but were mainly dependent on photoperiod and reproductive stage, whereas luteinizing hormone levels remained low throughout. In summary, dietary supplementation with 40 ppm (45.76 mg/kg) T4 was successful in inducing molt in turkey breeder hens. However, dropping the photoperiod was necessary to completely reset the reproductive system.