Cloning and functional analysis of the proximal promoter region of the three GnRH genes from the silver sea bream (Sparus sarba)

Gonadotropin-releasing hormone (GnRH) is a neuropeptide that plays a major role in releasing pituitary gonadotropin and controlling vertebrate reproduction. In this study, three GnRH cDNAs, GnRH-I (sbGnRH; 348 bp), GnRH-II (cGnRH-II; 557 bp), and GnRH-III (sGnRH; 483 bp), were cloned from the brain of the silver sea bream (Sparus sarba). In order to understand how the expression of the GnRH isoforms was regulated in the brain, the promoter of each gene was cloned and analyzed. We found regulatory motifs in the promoters that were conserved in the GnRH promoters of tilapia and zebrafish, suggesting that these motifs play a critical role in GnRH regulation. We performed functional analyses and examined tissue-specific expression for each GnRH promoter using EGFP reporter fusions in zebrafish. The GnRH-I promoter was active in the forebrain area, including the olfactory bulb-terminal nerve area and peripheral preoptic areas; the GnRH-II promoter was active in the midbrain; and the GnRH-III promoter was active in the olfactory bulb. These results show that the GnRH promoters of the silver sea bream GnRH genes exhibit tissue-specific activity.     

Ontogenic development of three GnRH systems in the brain of a pleuronectiform fish, barfin flounder

A pleuronectiform fish, the barfin flounder Verasper moseri, has three molecular forms of gonadotropin-releasing hormone (GnRH) in the brain, salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II) and seabream GnRH (sbGnRH). To elucidate the ontogenic origin of the neurons that produce these GnRH molecules, the development of three GnRH systems was examined by in situ hybridization and immunocytochemistry. Neuronal somata that express sGnRH mRNA were detected first in the vicinity of the olfactory epithelium 21 days after hatching (Day 21), and then in the transitional area between the olfactory nerve and olfactory bulb and the terminal nerve ganglion on Day 28. cGnRH-II mRNA-expressing neuronal somata were first identified in the midbrain tegmentum near the ventricle on Day 7. cGnRH-II-immunoreactive (ir) fibers were first found in the brain on Day 7. sbGnRH mRNA-expressing neuronal somata were first detected in the preoptic area on Day 42. sbGnRH-ir fibers were localized in the preoptic area-hypothalamus, and formed a distinctive bundle of axons projecting to the pituitary on Day 70. These results indicate that three forms of GnRH neurons have separate embryonic origins in the barfin flounder as in other perciform fish such as tilapia Oreochromis niloticus and red seabream Pagrus major: sGnRH, cGnRH-II and sbGnRH neurons derive from the olfactory placode, the midbrain tegmentum near the ventricle and the preoptic area, respectively.     

Characterization and localization of gonadotropin-releasing hormone in the brain and pituitary of the three-spined stickleback,Gasterosteus aculeatus

Radioimmunoassay (RIA) studies on highperformance liquid chromatography (HPLC) fractions of brain extracts of the three-spined stickleback, Gasterosteus aculeatus, provided evidence for at least two forms of gonadotropin-releasing hormone (GnRH). One form showed chromatographic and immunological properties similar to that of synthetic salmon GnRH (sGnRH). A second, unidentified form of GnRH eluted in the same position as chicken GnRH I (cGnRH-I); however, it did not cross-react in a cGnRH-I RIA. Furthermore, it cannot be excluded that chicken GnRH II (cGnRH-II) and maybe one other unidentified form are present in the stickleback. The distribution of GnRH in the brain of breeding adult male sticklebacks was studied by use of immunohistochemistry. Two antisera against sGnRH and antisera against mGnRH and cGnRH-II were applied on cryosections and visualized using the peroxidase-antiperoxidase method. Staining patterns were similar after incubations with all four antisera. Immunoreactive fibers were found in most parts of the brain. Three distinct groups of GnRH-immunoreactive perikarya were found in the nucleus olfactoretinalis, in the nucleus anterior periventricularis, and in the nucleus lateralis tuberis. Moreover, weakly stained cells occurred in a periventricular position in the midbrain. The proximal pars distalis of the pituitary, housing the gonadotropic cells, was richly innervated by GnRH-positive fibers. In the pars intermedia and in the rostral pars distalis, immunoreactive fibers were absent.     

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.     

Three GnRH systems in the brain and pituitary of a pleuronectiform fish,the barfin flounder Verasper moseri

To clarify the possible function of gonadotropin-releasing hormone (GnRH) in the brain of a pleuronectiform fish, the barfin flounder Verasper moseri, the distribution of three forms of GnRH in various areas of the brain was examined by radioimmunoassay, and the localization of GnRH-immunoreactive (ir) cell bodies and fibers in the brain and pituitary was determined by immunocytochemistry. The dominant form in the pituitary was seabream GnRH (sbGnRH), levels of which were much higher than those of salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II). In contrast, sbGnRH levels were extremely low in all other brain areas examined. Levels of sGnRH and cGnRH-II were high in the anterior and posterior part of the brain, respectively. sbGnRH-ir cell bodies were located in the preoptic area, whereas sbGnRH-ir fibers were localized mainly in the preoptic area-hypothalamus-pituitary and formed a distinctive bundle of axons projecting to the pituitary. sGnRH-ir cell bodies were located in the ventromedial part of the rostral olfactory bulbs and in the terminal nerve ganglion (the transitional area between the olfactory bulb and the telencephalon). cGnRH-II-ir cell bodies were localized to the midbrain tegmentum. sGnRH-ir and cGnRH-II-ir fibers were observed throughout the brain except in the pituitary gland. These results indicate that sbGnRH is responsible for the neural control of the reproductive endocrinology of the barfin flounder (hypothalamo-hypophysial system), and that sGnRH and cGnRH-II function as neurotransmitters or neuromodulators in the brain.     

Brain morphology and immunohistochemical localization of the gonadotropin-releasing hormone in the bluefin tuna, Thunnus thynnus

The present study was focused on the morphology of the diencephalic nuclei (likely involved in reproductive functions) as well as on the distribution of GnRH (gonadotropin-releasing hormone) in the rhinencephalon, telencephalon and the diencephalon of the brain of bluefin tuna (Thunnus thynnus) by means of immunohistochemistry. Bluefin tuna has an encephalization quotient (QE) similar to that of other large pelagic fish. Its brain exhibits well-developed optic tecta and corpus cerebelli. The diencephalic neuron cell bodies involved in reproductive functions are grouped in two main nuclei: the nucleus preopticus-periventricularis and the nucleus lateralis tuberis. The nucleus preopticus-periventricularis consists of the nucleus periventricularis and the nucleus preopticus consisting of a few sparse multipolar neurons in the rostral part and numerous cells closely packed and arranged in several layers in its aboral part. The nucleus lateralis tuberis is located in the ventral-lateral area of the diencephalon and is made up of a number of large multipolar neurones. Four different polyclonal primary antibodies against salmon (s)GnRH, chicken (c)GnRH-II (cGnRH-II 675, cGnRH-II 6) and sea bream (sb)GnRH were employed in the immunohistochemical experiments. No immunoreactive structures were found with anti sbGnRH serum. sGnRH and cGnRH-II antisera revealed immunoreactivity in the perikarya of the olfactory bulbs, preopticus-periventricular nucleus, oculomotor nucleus and midbrain tegmentum. The nucleus lateralis tuberis showed immunostaining only with anti-sGnRH serum. Nerve fibres immunoreactive to cGnRH and sGnRH sera were found in the olfactory bulbs, olfactory nerve and neurohypophysis. The significance of the distribution of the GnRH-immunoreactive neuronal structures is discussed.     

Gonadotropin releasing hormone (GnRH) neurons project to growth hormone and somatolactin cells in the steelhead trout

Analysis of gene expression using gonadotropin-releasing hormone (GnRH) antisense oligonucleotide confirmed by immunocytochemical localization the occurrence of GnRH neurons along the nervus terminalis in the steelhead trout (Oncorhynchus mykiss). Double-label immunocytochemistry revealed the distribution of mammalian (m), salmon (s) and chicken II (cII)-type GnRHs and various pituitary hormones. Both sGnRH and mGnRH appeared to be colocalized in the same cells of the nervus terminalis. Chicken GnRH II-immunoreactivity was found only in fibers and terminals. In the younger fish [73 and 186 days after fertilization (DAF)] GnRH neurons were seen rostral to the olfactory bulb. A novel GnRH ganglion, along the nervus terminalis, was found at the cribriform bone (gCB). A few non-immunoreactive rounded cells were seen among the GnRH neurons. A second smaller ganglion was seen at the most rostrally located part of the ventromedial olfactory bulb (gROB). In the older fish (850 DAF) GnRH neurons were also observed in the basal forebrain. A small group of neurons (2–3 cells), at the caudoventromedial border of the olfactory bulb, formed the ganglion terminale. Occasionally isolated GnRH-immunoreactive cells were seen at the base of the olfactory epithelium, along the ventromedial margins of the olfactory nerve. GnRH-immunoreactive and GnRH mRNA expressing neurons were absent from midbrain regions at the ages observed. GnRH-immunoreactive fibers were present only in older fish. The pattern of distribution of fibers that were immunoreactive to all three forms of GnRH was identical. Fibers were seen along the medial side of the olfactory nerve, throughout the brain and in the pituitary, associated with growth hormone and somatolactin cells. This morphological study shows that molecular forms of GnRHs might have multiple functions.     

Time- and dose-related effects of gonadotropin-releasing hormone on growth hormone and gonadotropin subunit gene expression in the goldfish pituitary

The goldfish brain contains two molecular forms of gonadotropin-releasing hormone (GnRH): salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II). In a preliminary report, we demonstrated the stimulation of gonadotropin hormone (GtH) subunit and growth hormone (GH) mRNA levels by a single dose of GnRH at a single time point in the goldfish pituitary. Here we extend the work and demonstrate time- and dose-related effects of sGnRH and cGnRH-II on GtH subunit and GH gene expression in vivo and in vitro. The present study demonstrates important differences between the time- and dose-related effects of sGnRH and cGnRH-II on GtH subunit and GH mRNA levels. Using primary cultures of dispersed pituitary cells, the minimal effective dose of cGnRH-II required to stimulate GtH subunit mRNA levels was found to be 10-fold lower than that of sGnRH. In addition, the magnitudes of the increases in GtH subunit and GH mRNA levels stimulated by cGnRH-II were found to be higher than the sGnRH-induced responses. However, no significant difference was observed between sGnRH and cGnRH-II-induced responses in vivo. Time-related studies also revealed significant differences between sGnRH- and cGnRH-II-induced production of GtH subunit and GH mRNA in the goldfish pituitary. In general, the present study provides novel information on time- and dose-related effects of sGnRH and cGnRH-II on GtH subunit and GH mRNA levels and provides a framework for further investigation of GnRH mechanisms of action in the goldfish pituitary.     

The effect of gonadotropin-releasing hormone on growth hormone and gonadotropin subunit gene expression in the pituitary of goldfish, Carassius auratus

The goldfish brain contains at least two forms of gonadotropin-releasing hormone (GnRH): sGnRH and cGnRH-II. In goldfish sGnRH and cGnRH-II are present both in the brain and pituitary, and exert direct effects via specific GnRH receptors stimulating growth hormone (GH) and gonadotropin hormone (GtH) synthesis and secretion. In this study, we investigated the effects of sGnRH and cGnRH-II on GtH subunit (alpha, FSH-beta and LH-beta) and GH mRNA levels in the goldfish pituitary in vivo and in vitro. Injection of goldfish with sGnRH or cGnRH-II (4 microg/fish) stimulated GtH-alpha, FSH-beta and LH-beta mRNA levels after 24 h. For in vitro studies, goldfish pituitary fragments were treated continuously for 12 h with 10(-7) M sGnRH or cGnRH-II. Both sGnRH and cGnRH-II stimulated GtH-alpha, FSH-beta, LH-beta and GH mRNA levels, however, cGnRH-II appeared to have a more pronounced effect. Similar experiments were carried out using cultured dispersed goldfish pituitary cells. In this study, treatments for 12 h with 10(-7) M sGnRH or cGnRH-II also stimulated GtH and GH gene expression. The present results provide a basis for the investigation of the signal transduction pathways that mediate GnRH-induced changes in GtH subunit and GH mRNA levels in the goldfish pituitary.     

Differential Distribution of Gonadotropin-Releasing Hormone Variants in the Brain of Hydrochaeris hydrochaeris (Mammalia, Rodentia)

1.In a previous paper we reported evidence for the presence of mGnRH- and sGnRH-like peptides in the preoptic–hypothalamic region of the capybara Hydrochaeris hydrochaeris (Montaner et al., 1998). In that study, the presence of a cGnRH-II like molecule in olfactory bulb extracts was suggested.2.The capybara, the largest living rodent in the world, belongs to the order Hystricomorpha, which is considered to be one of the oldest groups of rodents. Some authors consider that this group is the ancestor of all remaining rodents.3.In this study we have characterized GnRH molecular variants found in extracts from the olfactory bulbs and the mesencephalic region of capybara. These regions represent the two GnRH neuronal systems: the terminal nerve–septopreoptic and the midbrain systems.4.An indirect method combining reverse-phase high-performance liquid chromatography (RP-HPLC) and radioimmunoassay (RIA) was used to characterize GnRH variants. The analysis of both extracts with two different RIA systems revealed three immunoreactive GnRH peaks, coeluting with mGnRH, cIIGnRH, and sGnRH synthetic standards. These results were additionally supported by serial dilution studies with specific antisera.5.To our knowledge this the first report on the presence of three GnRH variants in the brain of an eutherian mammal. These results suggest that, similarly to other vertebrates, the expression of multiple GnRH variants may also be a common pattern in mammals.     

A novel form of gonadotropin-releasing hormone in the medaka, Oryzias latipes

The present study has identified three molecular forms of gonadotropin-releasing hormone (GnRH) in the brain of a teleost, the medaka, by isolation of their cDNAs. This species has a novel GnRH, which is here named medaka-type GnRH (mdGnRH), in addition to two characterized forms, chicken-II-type GnRH (cGnRH-II) and salmon-type GnRH (sGnRH). Phylogenetic analysis showed that mdGnRH is a medaka homolog of and seabream-type GnRH (sbGnRH) and mammalian-type GnRH (mGnRH) in other species, and suggested that all vertebrates have three distinct GnRHs. Furthermore, in situ hybridization revealed that the mdGnRH gene is expressed only in neurons clustered within the preoptic area as sbGnRH and mGnRH genes in other species are, while the genes for cGnRH-II and sGnRH are only in the midbrain tegmentum and nucleus olfactoretinalis, respectively. This result suggested that mdGnRH is a hypophysiotropic factor and the other two forms are involved in other physiological events as neuromodulators or neurotransmitters.     

Gonadotropin-releasing hormone (GnRH): from fish to mammalian brains

1. This work deals with a family of neuropeptides, gonadotropin-releasing hormone (GnRH), that play a key role in the development and maintenance of reproductive function in vertebrates.2. Until now, a total of 16 GnRH structural variants have been isolated and characterized from vertebrate and protochordate nervous tissue. All vertebrate species already investigated have at least two GnRH forms coexisting in the central nervous system. However, it is now well accepted that three forms of GnRH in early and late evolved bony fishes are present.3. In these cases, cGnRH-II is expressed by midbrain neurons, a species-specific GnRH is present mainly in the preoptic area and the hypothalamus, and sGnRH is localized in the terminal nerve ganglion (TNG). In this context it is possible to think that three GnRH forms and three GnRH receptor (GnRH-R) subtypes are expressed in the central nervous system of a given species.4. Then it is possible to propose three different GnRH lineages expressed by distinct brain areas in vertebrates: (1) the conserved cGnRH-II or mesencephalic lineage; or (2) the hypothalamic or releasing lineage whose primary structure has diverged by point mutations (mGnRH and its orthologous forms: hrGnRH, wfGnRH, cfGnRH, sbGnRH, and pjGnRH); and (3) the telencephalic sGnRH form. Also different GnRH nomenclatures are discussed.     

Cloning of FSH-β, LH-β and glycoprotein hormone α subunits in pejerrey Odontesthes bonariensis (Valenciennes): expression profile and relationship with GnRH expression and plasma sex steroid levels in male fish

Three cDNAs encoding pejerrey Odontesthes bonariensis follicle stimulating hormone-β (FSH-β), luteinizing hormone-β (LH-β) and glycoprotein-α (GPH-α) subunits were cloned and characterized. Gene expression of these subunits was analysed by real-time polymerase chain reaction (PCR) and compared with the brain gene expression of endogenous gonadotropin-releasing hormones (GnRHs): Pacific salmon GnRH (GnRH-III), pejerrey GnRH (GnRH-I) and chicken GnRH-II (GnRH-II) and plasma sex steroid levels in adult males. The nucleotide sequences of the FSH-β, LH-β and GPH-α subunits are 466, 558 and 677 base pairs long, encoding for mature peptides of 102, 118 and 98 amino acids respectively. Maturing males had high expression of FSH-β and GPH-α subunits, and intermediate levels of LH-β when compared with running ripe and spent stages. These animals had the lowest plasma testosterone (T) and 11-ketosterone (11-KT) values as well as low expression of sGnRH, cGnRH-II and pjGnRH. Running ripe males had the lowest expression of FSH-β and the highest expression of LH-β and GPH-α subunits, and of the three GnRH genes. At this stage, the highest values of T and 11-KT were observed. Spent males showed low expression of the three gonadotropin (GtH) subunits, sGnRH, pjGnRH and low levels of T. At this stage, 11-KT levels and cGnRH-II expression showed a tendency to decrease but the values were not statistically significant ( P < 0·05) to running ripe stage. The present results would suggest that T and 11-KT modulate the expression of the FSH subunits. The expression of the anterior brain GnRH variants, sGnRH and pjGnRH is correlated with LH-β expression and reinforce the importance of the forebrain GnRH variants on the regulation of pituitary function.