Ontogenic origin of salmon GnRH neurons in the ventral telencephalon and the preoptic area in masu salmon

During the ontogeny of masu salmon Oncorhynchus masou, neurons producing the salmon type of gonadotropin-releasing hormone (sGnRH) were first detected in the olfactory epithelium of the eyed egg and, subsequently, in the brain, suggesting a migration of these cells. Among sGnRH neurons distributed from the olfactory nerve (ON) through the preoptic area (POA), those in the ventral telencephalon (VT) and the POA are indicated to regulate gonadotropin secretion. Thus, it is of interest to know whether all the sGnRH neurons originate from the olfactory epithelium. In the present study, we examined by in situ hybridization whether sGnRH neurons are present in the VT-POA of fish, whose olfactory epithelia including sGnRH clusters were cauterized just after hatching (44 days after fertilization). Fish were sampled in June (212 days after the operation). Neurons expressing sGnRH mRNA were detected in the VT-POA as well as in the ON, ventral olfactory bulb, and transitional area between the olfactory bulb and telencephalon (which is considered to correspond to the terminal nerve ganglion) in the control group. In contrast, neurons expressing sGnRH mRNA were not detected in the VT-POA in the olfactory epithelium lesioned (OEL) group. Furthermore, pituitary sGnRH content in the OEL group was just above the detectable limit and was significantly lower than that in the corresponding control group in both sexes. These results indicate that sGnRH neurons in the VT-POA are derived from the olfactory epithelium in masu salmon, although the possibility cannot be ruled out that sGnRH neurons in the VT-POA arise from the VT-POA, but were delayed in expressing sGnRH because of the trauma of cauterization.     

Molecular cloning of three cDNAs encoding different GnRHs in the brain of barfin flounder

To examine the reproductive endocrinology of a large pleuronectiform fish, barfin flounder, Verasper moseri, a promising candidate for aquaculture and resource enhancement in northern Japan due to its high commercial value, three gonadotropin-releasing hormones (GnRHs) in the brain was identified by isolation of their cDNAs. This species had three molecular forms of GnRH; salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II), and seabream GnRH (sbGnRH). Each GnRH cDNA encoded a signal peptide (SP), GnRH, and a GnRH-associated peptide (GAP), which was connected to GnRH by a Gly-Lys-Arg sequence. The sGnRH cDNA encoded an SP composed of 23 amino acids and a GAP composed of 54 amino acids. The cGnRH-II cDNA encoded an SP of 23 amino acids and a GAP of 49 amino acids. The sbGnRH cDNA encoded an SP of 26 amino acids and a GAP of 57 amino acids. In situ hybridization showed that the genes for sGnRH, cGnRH-II, and sbGnRH are expressed in the ventromedial olfactory bulbs and the terminal nerve ganglion, the midbrain tegmentum, and the preoptic area, respectively. These results indicate that sbGnRH neurons in the preoptic area are involved in gonadotropin secretion in barfin flounder.     

Expression of GnRH genes is elevated in discrete brain loci of chum salmon before initiation of homing behavior and during spawning migration

Our previous studies suggested the importance of gonadotropin-releasing hormones (GnRHs) for initiation of spawning migration of chum salmon, although supporting evidence had been not available from oceanic fish. In farmed masu salmon, the amounts of salmon GnRH (sGnRH) mRNAs in the forebrain increased in the pre-pubertal stage from winter through spring, followed by a decrease toward summer. We thus hypothesized that gene expression for GnRHs in oceanic chum salmon changes similarly, and examined this hypothesis using brain samples from winter chum salmon in the Gulf of Alaska and summer fish in the Bering Sea. They were classified into sexually immature and maturing adults, which had maturing gonads and left the Bering Sea for the natal river by the end of summer. The absolute amounts of GnRH mRNAs were determined by real-time PCRs. The amounts of sGnRH mRNA in the maturing winter adults were significantly larger than those in the maturing summer adults. The amounts of sGnRH and chicken GnRH mRNAs then peaked during upstream migration from the coast to the natal hatchery. Such changes were observed in various brain loci including the olfactory bulb, terminal nerve, ventral telencephalon, nucleus preopticus parvocellularis anterioris, nucleus preopticus magnocellularis and midbrain tegmentum. These results suggest that sGnRH neurons change their activity for gonadal maturation prior to initiation of homing behavior from the Bering Sea. The present study provides the first evidence to support a possible involvement of neuropeptides in the onset of spawning migration.     

Effects of photoperiod on salmon GnRH mRNA levels in brain of castrated underyearling precocious male masu salmon.

Previous studies have suggested that activation of salmon gonadotropin-releasing hormone (sGnRH)-producing neurons is induced by the combined effects of photoperiod and steroid hormones in underyearling males of the masu salmon, Oncorhynchus masou. The present study further assesses the effects of photoperiod and steroid hormones on sGnRH synthetic activity and examines the changes in sGnRH mRNA levels in the brains of castrated underyearling precocious male masu salmon by manipulating the photoperiod for 60 days from August through October. In castrated males in which plasma testosterone levels decreased to low levels, sGnRH mRNA levels in the preoptic area (POA) increased under a short photoperiod (8L-16D), whereas they remained at low levels under a long photoperiod (16L-8D) for a 2-month duration. In sham-operated males, sGnRH mRNA levels in the ventral telencephalon and those in the POA increased in October with testicular maturation even under a long photoperiod with a delay of 1 month compared with the short photoperiod group. These results suggest that preoptic sGnRH-producing neurons receive short photoperiodic signals and that either short photoperiod or steroid hormone secretion is required for the activation of sGnRH synthesis in underyearling precocious male masu salmon.     

Activation of Salmon GnRH mRNA Expression Prior to Differentiation of Precocious Males in Masu Salmon

Changes in salmon gonadotropin-releasing hormone (sGnRH) mRNA levels in the brain of underyearling male masu salmon,Oncorhynchus masou,were investigated to clarify sGnRH participation in differentiation of precocious males. Fish were immature with low gonadosomatic index (GSI < 0.100%) on April 16 and May 7. On June 11, fish were differentiated into two groups: future precocious males and immature males. Fish in the former group had high GSI (0.203%) with proliferation of primary spermatocytes in the testis, while the latter had low GSI (0.055%) with primary spermatogonia. sGnRH mRNA levels (the number of neurons expressing sGnRH mRNA and the total number of silver grains per unit area) in the ventral telencephalon (VT) and in the VT plus preoptic area (POA) already increased in May before the differentiation of precocious males. GSI was positively correlated with sGnRH mRNA levels in the VT and in the VT plus POA in April, and with mRNA levels in the POA in May. Activated sGnRH synthesis is concluded to be related to the appearance of precocious males in masu salmon.     

The Atlantic salmon prepro-gonadotropin releasing hormone gene and mRNA.

Screening for the gene encoding salmon gonadotropin releasing hormone (sGnRH) in an Atlantic salmon (Salmo salar) genomic library resulted in isolation of a positive clone designated lambda sGnRH-1. An anchor polymerase chain reaction (PCR) technique was used to amplify GnRH cDNA derived from salmon hypothalamic mRNA. The cDNA sequence was aligned to the 7607 base pair genomic sequence which was shown to encode the entire prepro-GnRH gene. The cDNA proved that the cloned gene is expressed in the hypothalamus of mature salmon. The coding domain of sGnRH differs from the mammalian GnRH by six nucleotide changes which allow the two amino acid differences between the two GnRH variants. Salmon GnRH associated peptide (GAP) differs extensively in sequence and size from the mammalian counterpart. Compared to the GnRH cDNA of a cichlid species the similarity is 69.3% in the protein coding sequence.     

Two different messenger RNAs for salmon gonadotropin-releasing hormone are expressed in rainbow trout (Oncorhynchus mykiss) brain.

Two different precursor genes encoding the decapeptide salmon GnRH (sGnRH) are present in most salmonid species. In rainbow trout, a precedent Southern blot study revealed the existence of two different sGnRH genes and, recently, two different genes and their complementary DNAs that encode the identical peptide sGnRH were isolated from ovary and testis. Our study confirms the existence of two different mRNAs encoding sGnRH (sGnRH mRNA-I and sGnRH mRNA-II) in the brain of rainbow trout and, for the first time, full-length complementary DNA sequences are given. Central and peripheral distributions of the two messengers are described and seem to indicate different regulation of their expression. sGnRH mRNA-I is found essentially in the olfactory bulbs and telencephalon, whereas sGnRH mRNA-II is more widely expressed in the brain. Our observations allow speculation on the respective roles of two genes encoding the same decapeptide.     

Gonadotropin-releasing hormone-immunoreactive neurons and associated nicotinamide adenine dinucleotide phosphate-diaphorase-positive neurons in the brain of a teleost, Rhodeus amarus

Using combined nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHd) histochemistry and salmon gonadotropin-releasing hormone (sGnRH) immunocytochemistry, it is reported for the first time that possible potential contacts occur between the nitric oxide (NO)- and the GnRH-containing neurons in the brain of a freshwater teleost, Rhodeus amarus. GnRH-immunoreactive (ir) neurons were observed in the olfactory nerve (OLN), olfactory bulb (OB), medial olfactory tract (MOT), ventral telencephalon (VT), nucleus preopticus periventricularis (NPP), nucleus lateralis tuberis (NLT), and midbrain tegmentum (MT). Although NADPHd neurons were widely distributed in the brain, only those having an association with GnRH-ir neurons are described. Based on the nature of the association between the GnRH and the NADPHd neurons, the former were classified into three types. The Type I GnRH neurons were characterized by the presence of NADPHd-positive granules in the perikarya and processes and occurred in the OLN, OB, MOT, and VT. The Type II GnRH neurons, having soma-soma or soma-process contacts with the NADPHd neurons, were restricted to the MT; the long processes of NADPHd cells crossed over either the perikarya or the thick processes of GnRH cells. However, the Type III GnRH neurons, found in the NPP and NLT, did not show direct contact, but a few NADPHd fibers were present in the vicinity. The terminal-soma contacts in the olfactory system and the VT and the soma-soma contacts in the MT represent the sites of possible potential contacts indicating a direct NO involvement in GnRH function, although NO action by diffusion remains possible. NO may influence the NPP and NLT GnRH cells by diffusion only, since a direct contact was not observed.     

Differences in salmon GnRH and chicken GnRH-II contents in discrete brain areas of male and female rainbow trout according to age and stage of maturity

We have developed sensitive and specific radioimmunoassays (RIA) for salmon gonadotropin-releasing hormone (sGnRH) and chicken GnRH-II (cGnRH-II). Synthetic sGnRH and cGnRH-II(2-10) were conjugated to bovine serum albumin and injected into rabbits to raise specific antisera. The antiserum against sGnRH showed cross-reactivities of 1.58 and 0.08% for cGnRH-II and lamprey GnRH, respectively. The antiserum against cGnRH-II showed cross-reactivities of 0.05 and 0.01% for sGnRH and lamprey GnRH, respectively. Both antisera were observed not to cross-react with mammalian GnRH and cGnRH-I or other peptide hormones. Synthetic sGnRH and cGnRH-II were iodinated using the chloramine-T method. The iodinated GnRH was purified by HPLC using a reverse-phase C18 column. The RIA system was developed as a double antibody method. Brain extracts of rainbow trout showed displacement curves which were parallel to the sGnRH and cGnRH-II standards in each RIA. HPLC analysis followed by RIA has revealed that rainbow trout brain contains two types of GnRH: sGnRH and cGnRH-II. Total sGnRH content in the brain was about three-fold higher than that of cGnRH-II. In the olfactory bulbs, telencephalon, optic tectum-thalamus, hypothalamus, and pituitary, sGnRH content (per region) was higher than cGnRH-II content, whereas cerebellum and medulla oblongata contained much more cGnRH-II than sGnRH. sGnRH content in the optic tectum-thalamus and pituitary was the highest in 1-year-old immature fish and 3-year-old mature fish, respectively. Medulla oblongata showed the highest cGnRH-II content in all groups. sGnRH concentrations (per milligram of protein) were high in the pituitary and intermediate in the olfactory bulbs, hypothalamus, and telencephalon. In all groups, the cGnRH-II concentration was high in the medulla oblongata, whereas the concentration in the olfactory bulbs and pituitary gland was below the detectable limit in most individuals.     

Cell density and intracellular translocation of glucocorticoid receptor-immunoreactive neurons in the kokanee salmon (Oncorhynchus nerka kennerlyi) brain, with an emphasis on the olfactory system

This study tested the hypothesis that neurons in olfactory regions of the kokanee salmon brain contain glucocorticoid receptors. Distribution and neuronal number of glucocorticoid receptor-like immunoreactive (GRir) neurons were identified in the kokanee salmon brain using immunohistochemistry with an antibody to GR (polyclonal rabbit anti-human, dilution 1:1500; and monoclonal mouse, dilution 5 micrograms/ml). Distribution of GRir neurons similar to the mammalian pattern was observed in the brains of sexually immature (n = 8; 4 female and 4 male) as well as spawning (n = 8; 4 female and 4 male) salmon. Olfactory-related areas containing GRir positive neuronal bodies included the internal cell layer of the olfactory bulb, ventral-lateral and lateral parts of the dorsal telencephalon (homologue of the mammalian hippocampus), ventral area of the telencephalon (homologue of the mammalian amygdala), glomerulosus complex of the thalamus, the preoptic area, and inferior lobe of the hypothalamus. The pattern of GRir neuronal distribution in sexually immature and spawning fish was similar. However, spawning fish brains, compared to sexually immature brains, exhibited a significantly greater GRir neuronal number in several olfactory regions in paired immunohistochemical runs. There also were differences in intraneuronal location of GRir in olfactory regions, with staining being predominantly cytoplasmic in sexually immature fish but nuclear in spawning fish. These results are consistent with a role for cortisol in olfactory-mediated homing in kokanee salmon. Although GRir were identified in many nonolfactory regions, the focus of this study is on GRir present in brain regions involved in olfaction.     

Ontogeny of GnRH-like immunoreactive neuronal systems in the forebrain of the Indian major carp, Cirrhinus mrigala

GnRH immunoreactivity appeared in the medial olfactory placode very early in the development of Cirrhinus mrigala. The immunoreactive elements were divisible into distinct migratory and non-migratory components. The migratory component appeared as a patch of intensely immunoreactive cells located close to the olfactory epithelium in day 6 post-fertilization larvae. Subsequently, these neurons migrate caudally along the ventromedial aspect of the developing forebrain and enroute give rise to GnRH immunoreactive neurons in the (1) nervus terminalis located in ventral and caudal part of the olfactory bulb (day 8), and (2) basal telencephalon (day 9). The non-migratory GnRH immunoreactive component appeared in the olfactory placode of day 1 post-fertilization larvae. It consisted of few olfactory receptor neuron (ORN)-like cells with distinct flask-shaped somata, dendrites that communicate with the periphery and a single axon on the basal side; GnRH immunoreactivity was seen throughout the neuron. Considerable increase in the number of immunoreactive ORNs was encountered in day 2 post-fertilization larvae. On day 3, the dendrites of ORNs sprout bunches of apical cilia, while on the basal side the axonal outgrowths can be traced to the olfactory bulb. GnRH immunoreactive fibers were distributed in the olfactory nerve layer in the periphery of the bulb and glomeruli-like innervation was clearly established in 5 days old larvae. The innervation to the olfactory bulb showed a considerable increase in GnRH immunoreactivity in 9 and 19 days old larvae. However, GnRH immunoreactivity in non-migratory as well as migratory components gradually diminished and disappeared altogether by the age of 68 days. Results of the present study suggest that GnRH may serve a neurotransmitter role in the ORNs during early stages of development in C. mrigala.     

Forebrain gonadotropin-releasing hormone neuronal development: insights from transgenic medaka and the relevance to X-linked Kallmann syndrome

Neurons that synthesize and release GnRH are essential for the central regulation of reproduction. Evidence suggests that forebrain GnRH neurons originate in the olfactory placode and migrate to their final destinations, although this is still a matter of controversy. X-linked Kallmann syndrome (X-KS), characterized by failed gonadal function secondary to deficient gonadotropin secretion, is caused by a mutation in KAL1, which is suggested to regulate the migration of forebrain GnRH neurons. Because rodents lack Kal1 in their genome and have GnRH neurons scattered throughout their forebrain, the development of forebrain GnRH neurons and the pathogenesis of X-KS have been difficult to study. In the present study, we generated transgenic medaka that expressed green fluorescent protein under the control of the gnrh1 and gnrh3 promoters for analyzing forebrain GnRH neuronal development. Our data revealed the presence of the following four gnrh1 neuronal populations: an olfactory region-derived ventral preoptic population, a dorsal preoptic population that migrates from the dorsal telencephalon, a medial ventral telencephalic population that migrates from the anterior telencephalon, and a nonmigratory ventral hypothalamic population. We found that all forebrain gnrh3 neurons, extending from the terminal nerve ganglion to the anterior mesencephalon, arise from the olfactory region and that trigeminal ganglion neurons express gnrh3. Maternal gnrh3 expression was also observed in oocytes and early embryos. We subsequently identified a KAL1 ortholog and its paralogous form in the medaka. Consistent with the X-KS phenotype, antisense knockdown of the medaka KAL1 ortholog resulted in the disruption of forebrain GnRH neuronal migration. Thus, these transgenic medaka provide a useful model system for studying GnRH neuronal development and disorders of GnRH deficiency.     

Molecular characterization of the GnRH system in zebrafish (Danio rerio): cloning of chicken GnRH-II,adult brain expression patterns and pituitary content of salmon GnRH and chicken GnRH-II

The zebrafish has proven to be a model system with unparalleled utility in vertebrate genetic and developmental studies. Substantially less attention has been paid to the potential role that zebrafish can play in answering important questions of vertebrate reproductive endocrinology. As an initial step towards exploiting the advantages that the zebrafish model offers, we have characterized their gonadotropin-releasing hormone (GnRH) system at the molecular level. GnRHs comprise a family of highly conserved decapeptide neurohormones widely recognized to orchestrate the hormonal control of reproduction in all vertebrates. We have isolated the gene and cDNA encoding chicken GnRH-II (cGnRH-II) from zebrafish, as well as several kilobases of upstream promoter sequence for this gene. As the gene encoding salmon GnRH (sGnRH) has been previously isolated (Torgersen et al, 2002), this is the second GnRH gene isolated from zebrafish to date. We have localized expression of these two genes in the brains of reproductively mature zebrafish using in situ hybridization. sGnRH is localized to the olfactory bulb-terminal nerve region (OB-TN), the ventral telencephalon-preoptic area (VT-POA) and, as we report here for the first time in any teleost species, the hindbrain. cGnRH-II is expressed exclusively in the midbrain, as has been found in all other jawed vertebrate species examined. Finally, the levels of both GnRH peptides in pituitaries of reproductively mature zebrafish were quantified using specific ELISAs. sGnRH pituitary peptide levels were shown to be 3- to 4-fold higher than cGnRH-II pituitary peptide. The cumulative results of these experiments allow us to conclude that zebrafish express just two forms of GnRH in a site-specific manner within the brain, and that sGnRH is the hypophysiotropic GnRH form. This work lays the foundation for further research into the control of reproduction in zebrafish, such as the functional significance of multiple GnRHs in vertebrates, and the molecular mechanisms controlling tissue-specific GnRH expression.     

Beta-endorphin-like immunoreactivity in the forebrain and pituitary of the teleost Clarias batrachus (Linn.)

The organization of beta-endorphin-like immunoreactivity in the olfactory system, forebrain, and pituitary of the teleost Clarias batrachus was investigated. Immunoreactivity was prominently seen in the sensory neurons and basal cells in the olfactory epithelium and in some cells in the periphery and center (granule cells) of the olfactory bulb. Immunoreactive fibers in the olfactory nerve enter the olfactory nerve layer of the olfactory bulb and branch profusely to form tufts organized as spherical neuropils in the glomerular layer. While fascicles of immunoreactive fibers were seen in the medial olfactory tracts, the lateral olfactory tracts showed individual immunoreactive fibers. Immunoreactive fibers in the medial olfactory tract extend into the telencephalon and form terminal fields in discrete telencephalic and preoptic areas; some immunoreactive fibers decussate in the anterior commissure, while others pass into the thalamus. While neurons of the nucleus lateralis tuberis revealed weak immunoreactivity, densely staining somata were seen at discrete sites along the wall of the third ventricle. Although a large population of immunoreactive cells was seen in the pars intermedia of the pituitary gland, few were seen in the rostral pars distalis and proximal pars distalis; immunoreactive fibers were seen throughout the pituitary gland.     

Development and differentiation of gonadotropin hormone-releasing hormone neuronal systems and testes in the Japanese eel (Anguilla japonica).

In this study we investigated the relationship between the development of the olfactory, preoptic, and midbrain gonadotropin-releasing hormone (GnRH) neuronal systems and testicular differentiation in eels (Anguilla japonica) from embryonic stages through adulthood (5.4-50 cm body length). GnRH-synthesizing neuronal populations were first observed in the youngest fish ( approximately 5.0 cm) at the rostrobasal and caudalmost olfactory bulbs immunoreactive to a "promiscuous" (nonspecific) GnRH antiserum (635.5), and in the preoptic area and midbrain tegmentum immunoreactive to chicken GnRH II antiserum. The eel brains lacked salmon and seabream GnRH immunoreactivity. The evidence from our study suggests that the olfactory, preoptic, and midbrain GnRH populations have origins independent from those of proliferative periventricular zones within the brain. However, the olfactory GnRH neurons could have migrated out of the olfactory placodes during ages earlier than those observed in this study. Although all three GnRH neuronal populations contribute to pituitary innervation to some degree, the preoptic GnRH innervation was pronounced in the pituitary when primordial germ cells (animals approximately 5.0 cm) differentiated into male germ cells (animals 14-16 cm) and, therefore, an association can be assumed between preoptic GnRH expression and testicular differentiation in the Japanese eel.     

Immunohistochemical demonstration of the presence and localization of diverse molecular forms of gonadotropin-releasing hormone in the lizard (Podarcis s. sicula) brain.

The immunohistochemical presence and the distribution pattern of four different molecular forms of gonadotropin-releasing hormone (GnRH) were investigated in the brain of both sexes of the lizard, Podarcis s. sicula. Animals used in this study were collected in November and April, representing two different periods of the reproductive cycle. The antisera used were those raised against synthetic mammalian GnRH, chicken GnRH-I and II, and salmon GnRH. Strong immunoreaction was obtained for salmon, chicken-I, and chicken-II GnRHs, whereas a very weak reaction was seen for the mammalian form of GnRH. The distribution of immunoreactive-GnRH perikarya and fibers did not vary with the sex, the reproductive condition of the animals, or the antiserum used. Also, the intensity of immunoreaction with any one antiserum was quite similar in both periods of the year and in all brains examined. The immunoreactive perikarya was seen as two distinct groups, one in the mesencephalon and the other in the infundibulum. Immunoreactive fiber endings were seen in the telencephalon, the optic tectum, the anterior preoptic area, the median eminence, the central grey matter, the rhombencephalon, and the cerebellum. No immunoreactive perikarya were seen in the telencephalon or the anterior preoptic area.