Signal transduction pathways and transcription factors involved in the gonadotropin-releasing hormone-stimulated gonadotropin subunit gene expression.

Gonadotropin-releasing hormone (GnRH) stimulates gonadotropin (GTH) subunit gene expression via G protein-coupled membrane receptors. GnRH-stimulated GTH subunit gene expression is mediated by protein kinase C (PKC) and Ca(2+) signaling pathways. Recent numerous reports on signal transduction pathways which are involved in GnRH stimulation of mammalian GTH subunit genes showed differential sensitivity of GTH subunit genes to the two signaling pathways. Our recent studies on salmon GTH (sGTH) IIbeta subunit gene showed that its stimulation by GnRH is dependent on the PKC pathway. Furthermore, gel retardation and mutagenesis studies suggested that pituitary homeo box 1 (Ptx1) and Sp1 mediate the GnRH-induced PKC signaling on the sGTHIIbeta gene. However, both PKC and Ca(2+) pathways are involved in the GnRH-stimulated GTH alpha and LHbeta genes. Different preference to the pathways were often reported in a certain GTH subunit gene in different circumstances, suggesting that molecular targets of the two signaling pathways are different. Ets-related factor and cAMP response element binding protein have been proposed as targets of GnRH signaling on GTH alpha genes. Sp1 and early growth response protein 1 play pivotal roles in GnRH-stimulated LHbeta gene expression in synergism with steroidogenic factor-1 and Ptx1. Activating protein-1 mediates GnRH-induced PKC signaling to stimulate FSHbeta gene expression. Therefore, divergent transcription factors are involved in GnRH stimulation of GTH subunit gene expression, and molecular mechanisms of GnRH stimulation may be partially conserved between sGTH IIbeta and mammalian LHbeta genes.

Prepubertal increases in the levels of two salmon gonadotropin-releasing hormone mRNAs in the ventral telencephalon and preoptic area of masu salmon.

Ontogenic changes in the expression levels of two salmon gonadotropin-releasing hormone genes (sGnRH-I and -II) were examined in the forebrain region including the ventral telencephalon and preoptic area of masu salmon by competitive reverse transcription-polymerase chain reaction (RT-PCR). Two genes showed similar expression patterns throughout the lifetime in both sexes, although the levels of sGnRH-II mRNA were about 20 times higher than those of sGnRH-I mRNA. In males, the levels of sGnRH mRNAs increased at the beginning of the second year and reached their maximum in the autumn. The levels decreased gradually until the autumn of the third year when fish sexually matured. In females, the levels reached their maximum in the first autumn and fluctuated considerably along with the seasons in the third year. These results suggest that, in the salmon brain, sGnRH genes are activated long before the sexual maturation under sexually different control mechanisms.

Effects of gonadotropin-releasing hormone analog on expression of genes encoding the growth hormone/prolactin/somatolactin family and a pituitary-specific transcription factor in the pituitaries of prespawning sockeye salmon.

Gonadotropin-releasing hormone (GnRH) is a possible secretagogue of growth hormone (GH) and somatolactin (SL) in teleosts. Effects of GnRH on the levels of pituitary mRNAs encoding GH, prolactin (PRL), and SL were therefore examined in prespawning sockeye salmon (Oncorhynchus nerka). A capsule of GnRH analog (GnRHa) was implanted into the dorsal muscle of maturing sockeye salmon for 3 weeks. The levels of hormonal mRNAs were then determined by a quantitative dot blot analysis using single-stranded sense DNA of the same sequence of mRNA as the standard. Further, we analyzed effects of GnRHa on expression of the genes encoding pituitary-specific transcription factor (Pit-1/GHF-1). Relative levels of Pit-1/GHF-1 mRNAs were estimated by Northern blot analysis, which showed specific 2- and 3-kb bands of mRNAs. GnRHa significantly increased the level of SL mRNA in the males, but not in the females, compared to the control fish. It did not induce significant increases in the levels of GH and PRL mRNAs in both the males and the females. The levels of Pit-1/GHF-1 mRNAs in the control males tended to be higher than those in the initial controls, so that GnRHa might not be effective in enhancing expression of Pit-1/GHF-1 gene, except for the level of 3-kb Pit-1/GHF-1 mRNA in the females treated with 150 microg GnRHa. The pattern of changes in the levels of Pit-1/GHF-1 mRNAs were similar to those of GH and PRL mRNAs in both the males and the females and to that of SL mRNA in the females. These results indicate that, in prespawning sockeye salmon, GnRH can stimulate SL gene expression, but probably not through the Pit-1/GHF-1-dependent system.

Seasonal changes in expression of neurohypophysial hormone genes in the preoptic nucleus of immature female masu salmon.

In relevance to osmoregulatory and reproductive functions, activity of the hypothalamic magnocellular neurosecretory system may vary seasonally in teleosts. The changes in the expression of vasotocin (VT) and isotocin (IT) genes were thus studied by an in situ hybridization technique and an immunohistochemical avidin-biotin complex method in immature female masu salmon (Oncorhynchus masou). The plasma levels of testosterone and estradiol were also measured by enzyme immunoassay. Fish were sampled in March, May, August, and November 1994 and January 1995. The intensity of autoradiographic hybridization signals and immunoreactivity were determined in individual neurosecretory cells (NSC) in the rostroventral, middle, and dorsocaudal regions of the magnocellular part of the preoptic nucleus (PM). The VT hybridization signals and immunoreactivity were high in November, along with the elevation of plasma levels of testosterone and estradiol. These results suggest that sex steroid hormones are involved in seasonal regulation of VT gene expression. The hybridization signals for IT mRNA were increased in May and decreased in November, whereas IT immunoreactivity was low in March and high in November. NSCs thus showed seasonal variations in the intensity of hybridization signals for VT and IT mRNAs and immunoreactivity of VT and IT, although the patterns of changes were different between VT and IT. VT and IT genes may be seasonally expressed under different regulatory mechanisms.

Differences in seasonal expression of neurohypophysial hormone genes in ordinary and precocious male masu salmon.

Our previous study showed the seasonal variations in expression of vasotocin (VT) and isotocin (IT) genes in preoptic magnocellular neurons of female masu salmon (Oncorhynchus masou). The changes in the level of VT mRNA were coincident with those in plasma testosterone and estradiol levels. In the present study, generality of this phenomenon in salmonid was verified in males. We examined changes in expression of VT and IT genes by an in situ hybridization technique and an immunohistochemical avidin-biotin complex method in the preoptic nuclei of ordinary and precocious male masu salmon. Plasma levels of testosterone and estradiol were measured by enzyme immunoassay. Fish were sampled in March, May, August, and November 1994 and January 1995. The intensities of hybridization signals for VT and IT mRNAs, as well as immunoreactivity of VT and IT, showed seasonal variations, although the profiles were different between the ordinary and precocious males. In the ordinary males, the intensities of hybridization signals for VT and IT mRNAs were high in January. These strong hybridization signals, representing elevation of VT and IT gene expression, were accompanied by increases in plasma levels of testosterone and estradiol. However, in precocious males, changes in VT and IT mRNA levels were not coincident with variation of plasma levels of sex steroid hormones. The sensitivity to sex steroid hormones of VT and IT gene expression may be different between the ordinary and precocious male masu salmon.

cDNA for ribosomal protein S2 in sockeye salmon, Oncorhynchus nerka.

We isolated a cDNA encoding ribosomal protein S2 in sockeye salmon, Oncorhynchus nerka, using a reverse transcriptase-polymerase chain reaction (RT-PCR) method. The cDNA encoding ribosomal protein S2 is composed of 933 nucleotides, and has a 5'-noncoding sequence of 9 bases, a 885 base open reading frame coding for a 294 amino acid polypeptide, and a 39 base 3'noncoding sequence. The amino acid sequence of sockeye salmon S2 protein deduced from the nucleotide sequence is highly homologous to those from the rat (86.1%) and Drosophila melanogaster (73.6%). The N-terminal region of S2 protein is rich in arginine-glycine sites, including eight tandem repeats, and has two consecutive copies of the RGGF motif. The sequences are considered to be requisites for nucleolar localization and binding to RNA for nucleolar proteins. Southern blot analysis indicates that there may be only a single copy of the S2 gene, which is a multiple copy gene in the rat and the fruit fly. Northern blot analysis shows that the S2 gene is expressed in the brain, pituitary, heart, liver kidney, muscle, testis and ovary of sockeye salmon.

Cytophysiology of gonadotropin-releasing-hormone neurons in chum salmon (Oncorhynchus keta) forebrain before and after upstream migration.

Cytophysiology of gonadotropin-releasing-hormone neurons in chum salmon (Oncorhynchus keta) was examined before and after upstream migration by an immunocytochemical technique with a specific antiserum to salmon gonadotropin-releasing hormone and an in situ hybridization technique with an oligonucleotide encoding salmon gonadotropin-releasing hormone precursor (pro-salmon gonadotropin- releasing hormone). In the forebrain (olfactory nerve, olfactory bulb, telencephalon, and preoptic area), salmon gonadotropin-releasing hormone-immunoreactive neurons and neurons showing signals for pro-salmon gonadotropin-releasing hormone mRNA were compared between fish from the coastal sea and those from the spawning ground. Neurons in the dorsal region of the olfactory nerve and in the ventral region of the transitional area between olfactory nerve and olfactory bulb showed strong salmon gonadotropin-releasing-hormone immunoreactivity and strong hybridization signals in fish from the coastal sea, but these activities and signals were not observed or were decreased in number in fish from the spawning ground. The neurons in the olfactory bulb, telencephalon, and preoptic area consistently revealed salmon gonadotropin-releasing-hormone immunoreactivity and hybridization signals, and the hybridization signals of salmon gonadotropin-releasing hormone in the telencephalon and the preoptic area were stronger in fish from the spawning ground than in those from the coastal sea. These findings suggest that salmon gonadotropin-releasing-hormone neurons in the olfactory nerve and the transitional area between olfactory nerve and olfactory bulb have different patterns of hormone production than those in the telencephalon and the preoptic area.

Two differing precursor genes for the salmon-type gonadotropin-releasing hormone exist in salmonids.

Salmon gonadotropin-releasing hormone (sGnRH) is considered to have an important role in the control of reproduction in salmonid fish. As a basis for understanding the physiological functioning of sGnRH at the molecular level, we characterized the nucleotide sequences of two types of cDNAs encoding the precursors of sGnRH in sockeye salmon (ss), Oncorhynchus nerka, by a cloning strategy based on reverse transcription-PCR. The two types of cDNAs are referred to as ss-pro-sGnRH-I and -II, and consisted of 435 and 481 bases respectively. Both precursors are predicted to contain a signal peptide, the hormone and a GnRH-associated peptide that is attached to the hormone via a Gly-Lys-Arg sequence. The presence of two types of mRNAs hybridizing with either cDNA was confirmed by Northern blot analysis of brain RNA from sockeye salmon, masu salmon, O. masou, and rainbow trout, O. mykiss. The ss-pro-sGnRH-I cDNA had 97.2% and 82.8% overall identity with sGnRH cDNA from masu salmon and putative sGnRH cDNA deduced from the gene of the Atlantic salmon, Salmo salar respectively, whereas the ss-pro-sGnRH-II cDNA had 80.0% and 91.2% overall identity with the former and the latter respectively. The nucleotide sequences of ss-sGnRH-I and -II cDNAs showed less similarity (79.3%). These results indicated that each salmonid species possesses two differing sGnRH genes. The results of Southern blot analysis using genomic DNA extracted from individuals support this evidence in sockeye salmon, masu salmon and rainbow trout.

Salmon GnRH synthesis in the preoptic area and the ventral telencephalon is activated during gonadal maturation in female Masu Salmon.

Changes in salmon gonadotropin-releasing hormone (sGnRH) synthetic activity in the brain during gonadal maturation were examined by in situ hybridization in 2-year-old female masu salmon. Oncorhynchus masou. During gonadal maturation, the numbers of neurons expressing sGnRH mRNA increased in the preoptic area and the ventral telencephalon, but not in the olfactory bulbs and the terminal nerve ganglion. The numbers of silver grains per neuron also increased in the preoptic area and the ventral telencephalon. These results indicate that sGnRH has multiple physiological functions according to the location of the neurons in the brain; neurons in the preoptic area and the ventral telencephalon are involved in gonadal maturation possibly by stimulating gonadotropin synthesis and release, whereas neurons in the olfactory bulbs and the terminal nerve ganglion may have different roles.

Short photoperiod accelerates preoptic and ventral telencephalic salmon GnRH synthesis and precocious maturation in underyearling male masu salmon.

The temporal relationship between testicular maturation and salmon gonadotropin-releasing hormone (sGnRH) mRNA expression was investigated in underyearling precocious male masu salmon, Oncorhynchus masou. Testicular maturation could be experimentally manipulated by changing the length of the light-dark photoperiod; maturation was accelerated in the short photoperiod group (8L-16D) and delayed in the long photoperiod group (16-8D). sGnRH mRNA and total silver grains in these loci in individual fish, increased with advancing testicular maturation. They were maximal in the short photoperiod group in August and in the long photoperiod group in September, when spermiation occurred. In contrast, marked changes in sGnRH synthetic activity in relation to testicular maturation were not observed in the terminal nerve ganglion or in the olfactory bulbs. sGnRH neurons in the preoptic area and the ventral telencephalon are clearly influenced by photoperiod and are involved in the control of gonadal maturation probably via gonadotropin secretion.

Activation of salmon gonadotropin-releasing hormone synthesis by 17 alpha-methyltestosterone administration in yearling masu salmon, Oncorhynchus masou.

Juvenile salmonid pituitary gonadotropin (GTH) contents are elevated after steroid hormone treatment, but the involvement of gonadotropin-releasing hormone (GnRH) is unclear. Activation of salmon GnRH (sGnRH) synthesis by 17 alpha-methyltestosterone (MT) administration has been examined in the brain of yearling masu salmon (future precocious males and immature females) using an in situ hybridization technique combined with radioimmunoassay. Oral MT application markedly increased pituitary GTH II beta, but not GTH I beta, contents in both sexes. In future precocious males, MT treatment increased the number of cells expressing sGnRH mRNA in the preoptic area about threefold, whereas there were no significant differences in the olfactory bulbs and the ventral telencephalon. No significant changes were observed in cell sizes nor the numbers of silver grain per 100 microns2 cell in any of the brain regions. Thus, in future precocious males, preoptic sGnRH neurons may be activated by sex steroids. In contrast, no significant changes were observed in sGnRH mRNA levels of immature females after MT treatment. These differences in responses to sex steroids of sGnRH cells in the preoptic area between future precocious males and immature females suggest that MT has indirect actions via sGnRH and/or direct actions on the pituitary in the former, and that MT acts directly on the pituitary in the latter.

Divergence of gene expression in neurohypophysial hormone precursors among salmonids.

Salmonid fish have pairs of genes for various hypothalamic and pituitary hormones including neurohypophysial hormones, vasotocin, and isotocin, probably because they are tetraploid. The problem here is whether two genes for the same hormone are expressed equally or differently. We therefore examined expression patterns of vasotocin and isotocin genes in four salmonid species using Northern blot analysis with chum salmon cDNAs as hybridization probes. The presence of two vasotocin and also two isotocin genes was confirmed by Southern blot analysis in rainbow trout and sockeye salmon which were not examined previously. Prior to Northern blot analysis, isotocin-I cDNA of sockeye salmon was determined and compared to those of chum salmon and masu salmon, since molecular probes are so specific that cross-species hybridization often leads misinterpretation in a quantitative study. The nucleotide sequence of sockeye salmon isotocin-I cDNA showed sufficiently high homology (> 96.0%) to those of chum salmon and masu salmon for cross-species hybridization among salmonids. Northern blot analysis showed that both isotocin-I and isotocin-II genes were well expressed in all species examined. Expression of isotocin-I gene tended to be relatively higher than that of isotocin-II gene in all species. However, expression pattern of vasotocin-I and vasotocin-II genes did not coincide among species. Expression of vasotocin-II genes was very weak or scarce in masu salmon and rainbow trout, while that in sockeye salmon was stronger than vasotocin-I gene expression. The present result may reflect complicated molecular evolution of salmonid vasotocin genes probably both in regulatory and coding regions.

Characterization and localization of mRNA encoding the salmon-type gonadotrophin-releasing hormone precursor of the masu salmon.

Gonadotrophin-releasing hormone (GnRH) is considered to have an important role in the control of reproduction in salmonid fish, although we do not have any direct evidence. To clarify this problem by molecular techniques, we first determined the nucleotide sequence of the mRNA encoding the precursor of salmon-type GnRH (sGnRH) from the masu salmon, Oncorhynchus masou. The masu salmon sGnRH precursor was composed of a signal peptide, sGnRH and a GnRH-associated peptide (GAP) which was connected to sGnRH by a Gly-Lys-Arg sequence. The amino acid sequence of sGnRH and Gly-Lys-Arg were highly conserved when compared with the corresponding regions of African cichlid sGnRH and mammalian GnRH precursors. However, the GAP region was markedly divergent, with a 66% amino acid similarity to African cichlid GAP and an 8.3-15% similarity to mammalian GAPs. Northern blot analysis indicated the presence of a single mRNA species of about 600 bases in the olfactory bulb and telencephalon and in the diencephalon. The signal was more intense in the former regions. An in-situ hybridization study further revealed that sGnRH neurones were distributed in the olfactory nerve, the ventral part of the olfactory bulb, the ventral part of the telencephalon, the lateral preoptic area and the preoptic nucleus. The sGnRH neurones were thus longitudinally scattered between the olfactory nerve and the lateral preoptic area in the rostroventral part of brain. The intensity of the hybridization signals and the size of hybridization-positive somata were much greater in the olfactory nerve and the rostral olfactory bulb than in the other regions. Preoptic sGnRH neurones were scarcely detected in immature masu salmon, whereas they were more frequently observed in maturing animals. It is possible that the olfactory and the preoptic sGnRH neurones have different physiological roles in salmonid fish.

Effects of castration on volumes of the preoptic nucleus and the amygdala and on immunoreactivity of LH-RH fibers in the brain of the toad, Bufo japonicus.

We examined the influence of castration on the volumes of sexually dimorphic nuclei, the amygdala pars medialis (Am) and lateralis (Al), and the anterior part of the preoptic nucleus, and on the immunoreactivity of luteinizing hormone-releasing hormone (LH-RH) in brains of Japanese toads captured in spring and autumn. Animals were castrated (GnX) and half were implanted with testosterone (T) (GnX + T) and then killed and dissected after 1 month. Compared with sham-operated (Sham) toads, plasma androgen levels in autumn toads were significantly decreased by the castration, and those in both spring and autumn GnX toads were significantly elevated by the T implantation. The volume of Am in autumn toads was significantly reduced by GnX. Although not significant, changes in the volumes of the other nuclei, except for Al in spring toads, showed the following tendency 30 days after the operation: GnX + T greater than Sham greater than GnX. GnX did not alter LH-RH immunoreactivity in the median eminence. However, dense immunoreactive LH-RH fibers were found in the mesencephalic tegmental region in spring GnX toads but not in the other operation groups in both spring and autumn. LH-RH immunoreactivity was not altered in autumn toads. In spring GnX toads, thumb pads degenerated and evoked release calling was infrequent. These results suggest that (i) the volumes of sexually dimorphic nuclei, especially Am in the autumn toad, are androgen-dependent, and (ii) castration can modulate activity of the extrahypothalamic LH-RH-ergic projection in toad brain.

Extrahypothalamic projection of luteinizing hormone-releasing hormone fibers in the brain of the toad, Bufo japonicus.

Extrahypothalamic projection of luteinizing hormone-releasing hormone (LHRH) fibers in the brain of the toad (Bufo japonicus) was examined immunohistochemically by the avidin-biotin-peroxidase complex (ABC) method. Immunoreactive LHRH perikarya are localized in the nuclei medialis septi and of the diagonal band of Broca. A part of the LHRH fibers are sent anteriad to the medial and dorsal pallia. Some fibers reach the olfactory bulb. Dorsocaudally, LHRH neurons in the medial septum project their fibers to the deep layers of the optic tectum and the posterior mesencephalon including the nucleus pretrigeminalis, which is considered to be a generator of mate calling behavior, via the habenular and posterior thalamic regions. In addition, LHRH fibers which run caudad through the dorsal infundibular region and then the mesencephalic reticular formation were widely distributed in both the gray and the white matter of the medulla oblongata. These findings suggest that LHRH acts as a neurotransmitter or a neuromodulator in the various neuronal circuitries for reproductive behavior in the central nervous system, because LHRH has been considered to be related to amphibian seasonal breeding, and many regions where the immunoreactive LHRH fibers were observed are the loci concerned with mating behavior.

Projections of luteinizing hormone-releasing hormone and vasotocin fibers to the anterior part of the preoptic nucleus in the toad, Bufo japonicus.

The anterior part of the preoptic nucleus (APON) is a crucial locus for triggering male mate calling behavior in anuran amphibia. The projections to this locus of luteinizing hormone-releasing hormone and vasotocin fibers were immunohistochemically demonstrated in the brain of the toad (Bufo japonicus) using the avidin-biotin-peroxidase complex method. Immunoreactive (ir)-LH-RH perikarya are localized in the nucleus medialis septi and the nucleus of the diagonal band of Broca. A part of ir-LH-RH fibers arising from these nuclei project to the lateral part of the APON, where the APON neurons form their dendritic field, and often protrude into its medial neuronal cell mass. Meanwhile, a considerable number of vasotocin fibers arising from the ventral magnocellular part of the preoptic nucleus project anterior to these loci. These observations indicate that the LH-RH and vasotocin fibers may form ordinary and/or en passant synapses with the APON neurons to transmit peptidergic neuronal signals which are concerned with initiation of seasonal reproductive behavior.

An immunohistochemical study of seasonal changes in luteinizing hormone-releasing hormone and vasotocin in the forebrain and the neurohypophysis of the toad, Bufo japonicus.

Seasonal changes in luteinizing hormone-releasing hormone (LH-RH) and arginine vasotocin (AVT) were examined immunohistochemically in the toad forebrains and neurohypophyses. Strongly immunoreactive (ir-) LH-RH perikarya, from which dense ir-LH-RH fibers project to the median eminence, were localized in the medial septal nucleus and the nucleus of the diagonal band of Broca in the animals captured in the spring and the autumn. While, in the animals collected in the summer, ir-LH-RH perikarya and fibers were sparse, and immunoreactivity in the median eminences was weak. Artificially induced hibernation decreased the density of ir-LH-RH in the median eminence, in contrast with strong immunoreactivity in the control animals kept at room temperature. The amounts of ir-LH-RH in the median eminences of hibernating toads which were captured shortly before the breeding period varied conspicuously among individuals. The median eminences in migrating toads showed relatively weak LH-RH immunoreactivity. After the breeding, the immunoreactivity returned to the strong level that was observed in the spring and the autumn. These seasonal changes in ir-LH-RH seem to correspond to seasonal reproductive activity in this species. However, significant seasonal variations were not found in ir-AVT.