Distribution of salmon gonadotrophin releasing-hormone in the brain and pituitary of the sea bass (Dicentrarchus labrax)

Summary The distribution of salmon gonadotrophin-releasing hormone (sGnRH) was studied in the brain and pituitary of two-year-old immature sea bass (Dicentrarchus labrax) by means of an enzymoimmunoassay (EIA) for sGnRH and immunocytochemistry. The EIA for sGnRH is a competitive assay using a tracer made of sGnRH coupled to acetylcholinesterase from an electric eel. The separation of free and bound tracer is achieved by coating the plates with mouse anti-rabbit IgG monoclonal antibodies. Displacement curves generated by sGnRH and extracts from pituitary and different brain regions showed a good parallelism allowing the assay to be used for sGnRH measurements in this species. Although all parts of the brain contained measurable levels of sGnRH, the highest concentrations were found in the pituitary, the olfactory bulbs and the telencephalon. These data were confirmed by immunocytochemistry. Cell bodies were found in the olfactory bulbs, ventral telencephalon, preoptic region and mediobasal hypothalamus. Immunoreactive fibers could be observed in all parts of the brain including the optic tectum, the cerebellum (corpus and valvula), the vagal lobe, the medulla oblongata and the rostral spinal cord. In most cases, these fibers do not form well defined bundles; however, there was clearly a continuum of immunoreactive fibers, extending from the olfactory bulbs to the pituitary, and along which all the cell bodies described above were located. In the ventral telencephalon and the preoptic region, clear pictures of varicose positive fibers contacting immunoreactive perikarya could be observed. These data indicate that sGnRH is most likely an endogenous peptide in the brain of the sea bass, although the presence of other forms of GnRH cannot be excluded at this point. This study also demonstrates that the general organization of the GnRH systems in the sea bass is highly similar to what has been described in most freshwater teleost species, and provides basis for further studies on the neuroendocrine control of gonadotrophin release in this commercially important species.     

Ontogeny of the GnRH systems in zebrafish brain: in situ hybridization and promoter-reporter expression analyses in intact animals

The ontogeny of two gonadotropin-releasing-hormone (GnRH) systems, salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II), was investigated in zebrafish (Danio rerio). In situ hybridization (ISH) first detected sGnRH mRNA-expressing cells at 1 day post-fertilization (pf) anterior to the developing olfactory organs. Subsequently, cells were seen along the ventral olfactory organs and the olfactory bulbs, reaching the terminal nerve (TN) ganglion at 5–6 days pf. Some cells were detected passing posteriorly through the ventral telencephalon (10–25 days pf), and by 25–30 days pf, sGnRH cells were found in the hypothalamic/preoptic area. Continuous documentation in live zebrafish was achieved by a promoter-reporter expression system. The expression of enhanced green fluorescent protein (EGFP) driven by the sGnRH promoter allowed the earlier detection of cells and projections and the migration of sGnRH neurons. This expression system revealed that long leading processes, presumably axons, preceded the migration of the sGnRH neuron somata. cGnRH-II mRNA expressing cells were initially detected (1 day pf) by ISH analysis at lateral aspects of the midbrain and later on (starting at 5 days pf) at the midline of the midbrain tegmentum. Detection of red fluorescent protein (DsRed) driven by the cGnRH-II promoter confirmed the midbrain expression domain and identified specific hindbrain and forebrain cGnRH-II-cells that were not identified by ISH. The forebrain DsRed-expressing cells seemed to emerge from the same site as the sGnRH-EGFP-expressing cells, as revealed by co-injection of both constructs. These studies indicate that zebrafish TN and hypothalamic sGnRH cell populations share a common embryonic origin and migratory path, and that midbrain cGnRH-II cells originate within the midbrain. This study was supported by the US-Israel Bi-national Agricultural Research and Development (BARD) Foundation (grant 3428-03).     

Distribution of gonadotropin-releasing hormone immunoreactive systems in the brain of the Senegalese sole, Solea senegalensis

The present paper reports the immunohistochemical distribution of the gonadotropin-releasing hormone (GnRH) structures in the brain of the Senegalese sole, Solea senegalensis. In this study, we have used two antibodies against the salmon GnRH and chicken GnRH-II forms and the streptavidin–biotin-peroxidase complex method. Immunoreactive cell bodies are observed at the junction between the olfactory bulbs and the telencephalon (terminal nerve ganglion cells), in the ventral telencephalon, in the preoptic parvocellular nucleus, and in the synencephalic nucleus of the medial longitudinal fasciculus. GnRH-immunoreactive fibres were found extensively throughout the brain, located in the telencephalon, preoptic area, hypothalamus, hypophysis, optic tectum, midbrain and rhombencephalon. The antisera used in this study against the two GnRH forms exhibited cross-reactivity on the same cell masses and did not allow cell populations expressing different GnRH forms to be discriminated clearly. However, anti-salmon GnRH immunostained the GnRH cells and fibres of the forebrain much more intensely, whereas the anti-chicken GnRH antiserum shows a higher immunoreactivity on synencephalic cells of the medial longitudinal fasciculus.     

GnRH systems of <Emphasis Type="Italic">Cichlasoma dimerus</Emphasis> (Perciformes,Cichlidae) revisited: a localization study with antibodies and riboprobes to GnRH-associated peptides

The distribution of cells that express three prepro-gonadotropin-releasing hormones (GnRH), corresponding to salmon GnRH, sea bream GnRH (sbGnRH), and chicken II GnRH, was studied in the brain and pituitary of the South American cichlid fish, Cichlasoma dimerus. Although the ontogeny and distribution of GnRH neuronal systems have previously been examined immunohistochemically with antibodies and antisera against the various GnRH decapeptides, we have used antisera against various perciform GnRH-associated peptides (GAPs) and riboprobes to various perciform GnRH+GAPs. The results demonstrate that: (1) the GnRH neuronal populations in the forebrain (salmon and sea bream GAPs; sGAP and sbGAP, respectively) show an overlapping pattern along the olfactory bulbs, nucleus olfacto-retinalis, ventral telencephalon, and preoptic area; (2) projections with sGAP are mainly located in the forebrain and contribute to the pituitary innervation, with projections containing chicken GAP II being mainly distributed along the mid and hindbrain and not contributing to pituitary innervation, whereas sbGAP projections are restricted to the ventral forebrain, being the most important molecular form in relation to pituitary innervation; (3) sbGnRH (GnRH I) neurons have an olfactory origin; (4) GAP antibodies and GAP riboprobes are valuable tools for the study of various GnRH systems, by avoiding the cross-reactivity problems that occur when using GnRH antibodies and GnRH riboprobes alone.This work was supported by grants UBACYT X-217, Conicet PIP 0539/R188; NIH-HD-29186 and Fogarty International Fellowship TW00086.     

Development and use of striped bass-specific RFLP probes

We sought to develop nuclear DNA (nDNA) probes which could be used to complement mtDNA and DNA fingerprinting markers in distinguishing striped bass, Morone saxatilis (Walbaum), from discrete spawning systems. Restriction endonuclease-generated single copy, 10–20-kb striped bass nuclear nDNA fragments were cloned into the bacteriophage vector Lambda Dash II and tested in Southern blot analyses for their abilities to reveal population-specific polymorphisms. Three of the I7 nDNA sequences tested exhibited polymorphisms which potentially could be used to delineate striped bass populations. One probe, DSB 22, revealed significant genotypic frequency differences between Gulf of Mexico and Atlantic striped bass and among striped bass representative of some Atlantic systems. These preliminary results suggest that single copy nDNA sequences may provide sufficient polymorphisms to aid in stock identification of species which proved genetically monomorphic using other approaches.     

Isolation and characterization of 149 novel microsatellite DNA markers for striped bass,Morone saxatilis,and cross‐species amplification in white bass,Morone chrysops,and their hybrid

To support detailed genetic analysis of striped bass (Morone saxatilis) and white bass (Morone chrysops), we isolated 153 microsatellite loci from repeat‐enriched striped bass DNA libraries. Of these, 147 markers amplified in striped bass (average 4.7 alleles per locus) and 133 in white bass (average 2.2 alleles per locus). One hundred twenty‐two markers amplified in their hybrid. Development of new microsatellite markers will facilitate evaluations of genetic structure in wild populations and will support pedigree analysis and linkage mapping for selective breeding.     

Cloning and expression of gonadotropin-releasing hormone receptor in the brain and pituitary of the European sea bass: an in situ hybridization study

A full-length cDNA encoding a GnRH receptor (GnRH-R) has been obtained from the pituitary of the European sea bass, Dicentrarchus labrax. The complete cDNA is 1814 base pairs (bp) in length and encodes a protein of 416 amino acids. The 5' UTR and 3' UTR are 239 bp and 324 bp in size, respectively. The expression sites of this GnRH-R were studied in the brain and pituitary of sea bass by means of in situ hybridization. A quantitative analysis of the expression of the GnRH-R gene along the reproductive cycle was also performed. The GnRH-R brain expression was especially relevant in the ventral telencephalon and rostral preoptic area. Some GnRH-R messenger-expressing cells were also evident in the dorsal telencephalon, caudal preoptic area, ventral thalamus, and periventricular hypothalamus. A conspicuous and specific GnRH-R expression was detected in the pineal gland. The highest expression of the GnRH-R gene was observed in the proximal pars distalis of the pituitary. This expression was evident in all LH cells and some FSH cells but not in somatotrophs. In the pituitary, the quantitative analysis revealed a higher expression of GnRH-R gene during late vitellogenesis in comparison with maturation, spawning, and postspawning/resting periods. However, in the brain, the highest GnRH-R expression was evident at spawning or postspawning/ resting periods. These results suggest that the expression of this GnRH-R is regulated in a different manner in the brain and the pituitary of sea bass.     

Two differing salmon GnRH precursor mRNAs are co-expressed in the brain of sockeye salmon (Oncorhynchus nerka)

The localization of two salmon-type gonadotropin-releasing hormone (sGnRH) precursors, pro-sGnRH-I (short type) and pro-sGnRH-II (long type), was investigated by using in situ hybridization techniques in the brain of the landlocked sockeye salmon, Oncorhynchus nerka. We used 30-mer oligonucleotide probes complementary to pro-sGnRH-I and pro-sGnRH-II cDNA. No significant differences were observed in the localization of sGnRH neurons expressing pro-sGnRH-I and pro-sGnRH-II mRNAs; both were expressed in the olfactory nerve, the olfactory bulbs, the regions between the olfactory bulb and telencephalon, the ventral telencephalon, the preoptic area, and the hypothalamus. Almost all sGnRH neurons examined co-expressed both precursors. The expression of two sGnRH precursors in the same neuron and the wide distribution of such neurons in the brain suggest that there are no functional differences between the two precursors.     

Molecular mechanisms for migration of placodally derived GnRH neurons

Gonadotropin-releasing hormone (GnRH) neurons, critical for reproduction, are derived from the nasal placode and migrate into the brain along nasal axons. GnRH neurons appear to diverge from olfactory sensory cells during early stages of nasal placode differentiation. However, GnRH neurons rely on olfactory/vomeronasal axons as their pathway to the central nervous system (CNS). A novel factor, termed nasal embryonic luteinizing hormone-releasing hormone factor (NELF), was discovered in a differential screen of migrating versus nonmigrating GnRH neurons. NELF is expressed in olfactory sensory cells and GnRH cells in nasal areas. Antisense experiments demonstrated that knock-down of NELF decreased olfactory axon outgrowth and GnRH neuronal migration. These results indicate that NELF plays a role as a guidance molecule for olfactory axon projections and migration of GnRH cells. We hypothesize that NELF acts via a homophilic interaction and that NELF expression is critical for reproduction by insuring that GnRH cells reach the CNS. Furthermore, down-regulation of NELF on GnRH cells as they enter the telencephalon may allow GnRH cells to distinguish a different pathway(s) in the CNS (from those leading to olfactory regions) and thereby facilitate establishment of the appropriate adult-like GnRH distribution.     

Cytoarchitectonic study of the brain of a perciform species, the sea bass (Dicentrarchus labrax). I. The telencephalon

A cytoarchitectonic analysis of the telencephalon of the sea bass Dicentrarchus labrax, based on cresyl violet-stained serial transverse sections, is presented. Rostrally, the brain of the sea bass is occupied by sessile olfactory bulbs coupled to telencephalic hemispheres. The olfactory bulbs comprise an olfactory nerve fiber layer, a glomerular layer, an external cellular layer, a secondary olfactory fiber layer, and an internal cellular layer. Large terminal nerve ganglion cells are evident in the caudomedial olfactory bulbs. We recognized 22 distinct telencephalic nuclei which were classified in two main areas, the ventral telencephalon and the dorsal telencephalon. The ventral telencephalon displays four periventricular cell masses: the dorsal, ventral, supracommissural, and postcommissural nuclei; and four migrated populations: the lateral, central, intermediate, and entopeduncular nuclei. In addition, a periventricular cell population resembling the lateral septal organ reported in birds is observed in the ventral telencephalon of the sea bass. The dorsal telencephalon contains 13 nuclei, which can be organized into five major zones: the medial part, dorsal part, lateral part and its ventral, dorsal, and posterior divisions, the central part, and posterior part. Based on histological criteria, two cell masses are recognized in the ventral division of the lateral part of the dorsal telencephalon. The nucleus taenia is found in the caudal area of the dorsal telencephalon, close to the ventral area. This study represents a useful tool for the precise localization of the neuroendocrine territories and for the tracing of the neuronal systems participating in the regulation of reproduction and metabolism in this species.     

Use of cellular oncogene probes to identify Morone hybrids.

We tested the ability of cellular oncogene (c-onc) probes to identify F1 hybrids and the lineage of known backcrosses within the fish genus Morone. Total DNA was isolated from five to 14 individuals per North American Morone species (striped bass, white bass, white perch, and yellow bass). The DNA was digested with two restriction enzymes, Eco RI and Hin dIII, Southern blotted, and hybridized to six different c-onc probes including v-abl, v-erb B, c-myc, c-H-ras, c-K-ras, and v-src. We found fixed genotypic differences among the four species for all six probes in single restriction enzyme digests. The heritability of these nuclear DNA genotypes was evaluated in hatchery-produced F1 Morone hybrids (striped bass x white bass and striped bass x white perch) tested with the six informative single probe/restriction enzyme combinations. All F1 individuals exhibited heterozygosity in all diagnostic nuclear DNA fragments, confirming the Mendelian inheritance of these genotypes in these fish. Furthermore, analysis of these nuclear DNA genotypes in hatchery-produced backcrosses of F1 hybrids striped bass x (white bass x striped bass) detected both recombinant and parental genotypes at all six polymorphic c-onc sequences. The lineage of suspected Morone hybrids of unknown descent collected from Lewis Smith Lake, Alabama, and from the Occoquan River, Virginia, was determined using the c-onc probes. Our results suggest that c-onc probes are suitable markers to unequivocally identify F1 hybrids and backcrosses and to quantify introgression in natural populations of fishes. The addition of RFLP analysis of mtDNA provided a complete ancestral history of individual fish.     

Joint migration of gonadotropin-releasing hormone (GnRH) and neuropeptide Y (NPY) neurons from olfactory placode to central nervous system

The olfactory epithelium in vertebrates generates the olfactory sensory neurons and several migratory cell types. Prominent among the latter are the gonadotropin-releasing hormone (GnRH) neurons that differentiate within the olfactory epithelium during embryogenesis and migrate along the olfactory nerve to the central nervous system. We initiated studies to characterize additional neuronal phenotypes of olfactory epithelial derivation. Neuropeptide Y (NPY) neurons are functionally related to the reproductive axis, modulating the release of GnRH and directly enhancing GnRH-induced luteinizing hormone (LH) secretion from gonadotrophs. We demonstrate that a population of migratory NPY neurons originates within the olfactory epithelium of the chick. At stage 25, NPY-positive fibers, but not cells, were detected in the epithelium and the nerve. By stages 28–34, NPY neurons and processes were present in the olfactory epithelium, olfactory nerve, and at the junction of the olfactory nerve and forebrain. In these regions the number of NPY neurons increased until stage 30 and then declined as development progressed. Electron microscopic immunocytochemistry confirmed the neuronal phenotype of the NPY-positive cells. The origin and migratory nature of some of these NPY cells was confirmed by double-label immunocytochemical detection of NPY and GnRH. A large percentage of the NPY-cells coexpressed the GnRH peptide. Between stages 28 and 34 single- and double-labeled NPY and GnRH neurons were found side by side along the GnRH migratory route emanating from the nasal epithelium, along the olfactory nerve, and into the ventral forebrain. These data suggest that an NPY population originates in the olfactory epithelium and migrates into the central nervous system during embryogenesis. By stage 42, no NPY/GnRH double-labeled cells were detected. © 1996 John Wiley & Sons, Inc.     

Evolution of an MHC class Ia gene fragment in four North American Morone species

A nucleotide sequence analysis of a fragment of a Morone MHC class Ia gene detected high levels of polymorphism in striped bass Morone saxatilis, white perch Morone americana and yellow bass Morone mississippiensis. Extremely low levels of MHC diversity, however, were detected in white bass Morone chrysops, suggesting the possibility of a severe population bottleneck for this species.     

A reinvestigation of the Gn-RH (gonadotrophin-releasing hormone) systems in the goldfish brain using antibodies to salmon Gn-RH

Summary The organization of Gn-RH systems in the brain of teleosts has been investigated previously by immunohistochemistry using antibodies against the mammalian decapeptide which differs from the teleostean factor. Here, we report the distribution of immunoreactive Gn-RH in the brain of goldfish using antibodies against synthetic teleost peptide.Immunoreactive structures are found along a column extending from the rostral olfactory bulbs to the pituitary stalk. Cell bodies are observed within the olfactory nerves and bulbs, along the ventromedial telencephalon, the ventrolateral preoptic area and the latero-basal hypothalamus. Large perikarya are detected in the dorsal midbrain tegmentum, immediately caudal to the posterior commissure. A prominent pathway was traced from the cells located in the olfactory nerves through the medial olfactory tract and along all the perikarya described above to the pituitary stalk. In the pituitary, projections are restricted to the proximal pars distalis. A second immunoreactive pathway ascends more dorsally in the telencephalon and arches to the periventricular regions of the diencephalon. Part of this pathway forms a periventricular network in the dorsal and posterior hypothalamus, whereas other projections continue caudally to the medulla oblongata and the spinal cord. Lesions of the ventral preoptic area demonstrate that most of the fibers detected in the pituitary originate from the preoptic region.     

Isolation and Characterization of Microsatellite Loci for Striped Bass (Morone saxatilis)

Genetic variation has been difficult to detect in striped bass (Morone saxatilis). Therefore, we identified and characterized 13 microsatellite loci to provide additional genetic markers for striped bass. Microsatellites were identified by screening a striped bass genomic library or by using primers developed for European sea bass (Dicentrarchus labrax) microsatellite loci. We found that 6 of the 13 microsatellite loci were polymorphic in DNA samples obtained from wild populations of striped bass. The number of alleles per locus varied from 3 to 12, and the observed heterozygosities ranged from 0.55 to 0.78. These results indicate that microsatellite loci provide more alleles and higher heterozygosities than other genetic markers developed for striped bass. Received November 9, 1999; accepted February 11, 2000.     

Microsatellite DNA markers for parental assignment in hybrid striped bass (Morone saxatilis × Morone chrysops)

Development of nine polymorphic microsatellites from a genomic library of hybrid striped bass (female Morone chrysops × male Morone saxatilus) DNA is described. Breeding of hybrid striped bass for aquaculture is based largely on breeding wild fish. Molecular markers such as microsatellites will be useful tools for developing broodstock, estimating heritability for production traits, and selective breeding via marker‐assisted selection. The nine polymorphic microsatellites include six dinucleotide and three complex repeat motifs. The number of alleles detected among a sample of 10 individuals of each species was relatively low. All polymerase chain reaction primer pairs also amplified products in the sea bass Dicentrarchus labrax.     

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.