Takeshi Onuma

Year-to-year differences in plasma levels of steroid hormones in pre-spawning chum salmon.

Changes in plasma levels of steroid hormones in pre-spawning chum salmon (Oncorhynchus keta) were examined for 6 years in association with sexual maturation. Fish were sampled along their homing pathway from the coastal sea to the spawning ground from 1995 to 2000. Plasma levels of testosterone (T), 11-ketotestosterone (11KT), estradiol-17beta (E2), 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP), and cortisol were determined by enzyme immunoassays. Sexual maturity was comprehensively estimated by gonadosomatic indices, histology of gonads, nuptial color, spermiation or ovulation ratio. Since the plasma levels of steroid hormones and sexual maturation differed from year to year, they were compared with year-to-year variation of sea surface temperature (SST) of coastal sea to study influence of oceanographic environment on these physiological data. The SST of the migratory route varied among the years, so that we classified the 6 years into cool, intermediate, and warm years. Concerning maturity, the males that returned to the natal hatchery in the warm years were sexually more advanced than those in the cool years. Furthermore, histological data suggested that final oocyte maturation occurred before arrival at the hatchery in one of the warm years, i.e., 1999, while it occurred at the hatchery in one of the intermediate years, i.e., 2000. In the males, T and 11KT levels increased significantly on midway of the homing route in the warm years, whereas they did not show any noticeable changes in the cool years. Furthermore, the levels of T and 11KT on midway of the homing route in the warm years, i.e., 1998 and 1999, were significantly higher than those in one of the cool years, i.e., 1995, in both sexes. In the females, the levels of E2 decreased during upstream migration. Conversely, those of DHP considerably elevated at spawning ground in all years examined. The levels of cortisol were different from year to year regardless of the SST. The present results showed that there were year-to-year differences in plasma levels of steroid hormones and maturity, and some of them may be influenced by the year-to-year variation of SST.

Changes in expression of genes encoding gonadotropin subunits and growth hormone/prolactin/somatolactin family hormones during final maturation and freshwater adaptation in prespawning chum salmon

The pituitary levels of mRNAs encoding gonadotropin (GTH) subunits (GTH α2 and IIβ), prolactin (PRL), and somatolactin (SL) increased in chum salmon during the last stages of spawning migration. In the present study, changes in pituitary levels of mRNAs encoding GTH α2, Iβ, and IIβ; growth hormone (GH); PRL; and SL were examined in homing chum salmon of Sanriku stock to clarify whether the changes are associated with final maturation or freshwater (FW) adaptation. In 1993, fish were caught at four areas: off the coast of Sanriku (off-coast), the mouth of Otsuchi Bay (ocean), inside of Otsuchi Bay (bay), and the Otsuchi River (river). In addition, effects of hypoosmotic stimulation by transition from seawater (SW) to FW were examined in 1994 and 1995. The amounts of mRNAs were determined by dot-blot analyses or real-time polymerase chain reactions. The levels of GTH α2 and IIβ mRNAs in the ocean, bay, and river fish were two to five times those in the off-coast fish, and the levels of SL mRNAs in the bay fish were two to four times those in the off-coast fish. The levels of GH and PRL mRNAs in the ocean and bay fish were significantly lower than those in the off-coast fish, and those in the river fish were three to five times those in the ocean and bay fish. In the SW-to-FW transition experiment in 1994, the levels of GTH α2, Iβ, and IIβ mRNAs transiently increased, whereas changes were insignificant in 1995. The levels of GH, PRL, and SL mRNAs increased in both SW and FW environments, and no apparent effects of SW-to-FW transition were observed. The present study suggests that in prespawning chum salmon, expression of genes encoding GTH α2, IIβ, and SL elevates with final maturation regardless of osmotic environment. Hypoosmotic stimulation by transition from the SW-to-FW environment is not critical to modulate expression of genes for PRL. PRL gene expression can be elevated in SW fish that were sexually almost matured.

Stimulatory Effects of Insulin-Like Growth Factor 1 on Expression of Gonadotropin Subunit Genes and Release of Follicle-Stimulating Hormone and Luteinizing Hormone in Masu Salmon Pituitary Cells Early in Gametogenesis

Abstract Insulin-like growth factor-I (IGF-I) has been shown to be involved in pubertal activation of gonadot-ropin (GTH) secretion. The aim of this study was to determine if IGF-I directly stimulates synthesis and release of GTH at an early stage of gametogenesis. The effects of IGF-I on expression of genes encoding glycoprotein α (GPα), follicle-stimulating hormone (FSH) β, and luteinizing hormone (LH) β subunits and release of FSH and LH were examined using primary pituitary cells of masu salmon at three reproductive stages: early gametogenesis, maturing stage, and spawning. IGF-I alone or IGF-I + salmon GnRH (sGnRH) were added to the primary pituitary cell cultures. Amounts of GPα, FSHβ, and LHβ mRNAs were determined by real-time PCR. Plasma and medium levels of FSH and LH were determined by RIA. In males, IGF-I increased the amounts of all three subunit mRNAs early in gametogenesis in a dose-dependent manner, but not in the later stages. In females, IGF-I stimulated release of FSH and LH early in gametogenesis, whereas no stimulatory effects on the subunit mRNA levels were observed at any stage. IGF-I + sGnRH stimulated release of FSH and LH at all stages in both sexes, but had different effects on the subunit mRNA levels depending on subunit and stage. The present results suggest that IGF-I itself directly stimulates synthesis and release of GTH early in gametogenesis in masu salmon, possibly acting as a metabolic signal that triggers the onset of puberty.

Regulation of Temporal and Spatial Organization of Newborn GnRH Neurons by IGF Signaling in Zebrafish

When and how newborn neurons are organized to form a functional network in the developing brain remains poorly understood. An attractive model is the gonadotropin-releasing hormone (GnRH) neuron system, master regulator of the reproductive axis. Here we show that blockage of IGF signaling, a central growth-promoting signaling pathway, by the induced expression of a dominant-negative form of IGF1 receptor (IGF1R) or specific IGF1R inhibitors delayed the emergence of GnRH2 neurons in the midbrain and GnRH3 neurons in the olfactory bulb region. Blockage of IGF signaling also resulted in an abnormal appearance of GnRH3 neurons outside of the olfactory bulb region, although it did not change the locations of other olfactory neurons, GnRH2 neurons, or brain patterning. This IGF action is developmental stage-dependent because the blockade of IGF signaling in advanced embryos had no such effect. An application of phosphatidylinositol 3-kinase (PI3K) inhibitors phenocopied the IGF signaling deficient embryos, whereas the MAPK inhibitors had no effect, suggesting that this IGF action is mediated through the PI3K pathway. Real-time in vivo imaging studies revealed that the ectopic GnRH3 neurons emerged at the same time as the normal GnRH3 neurons in IGF-deficient embryos. Further experiments suggest that IGF signaling affects the spatial distribution of newborn GnRH3 neurons by influencing neural crest cell migration and/or differentiation. These results suggest that the IGF-IGF1R-PI3K pathway regulates the precise temporal and spatial organization of GnRH neurons in zebrafish and provides new insights into the regulation of GnRH neuron development.

Activity of the pituitary–gonadal axis is increased prior to the onset of spawning migration of chum salmon

SUMMARY The activity of the pituitary–gonadal axis (PG axis) in pre-migratory and homing chum salmon was examined because endocrine mechanisms underlying the onset of spawning migration remain unknown. Pre-migratory fish were caught in the central Bering Sea in June, July and September 2001, 2002 and 2003, and in the Gulf of Alaska in February 2006. They were classified into immature and maturing adults on the basis of gonadal development. The maturing adults commenced spawning migration to coastal areas by the end of summer, because almost all fish in the Bering Sea were immature in September. In the pituitaries of maturing adults, the copy numbers of FSHβ mRNA and the FSH content were 2.5- to 100-fold those of the immature fish. Similarly, the amounts of LHβ mRNA and LH content in the maturing adults were 100- to 1000-fold those of immature fish. The plasma levels of testosterone, 11-ketotestosterone and estradiol were higher than 10 nmol l–1 in maturing adults, but lower than 1.0 nmol l–1 in immature fish. The increase in the activity of the PG-axis components had already initiated in the maturing adults while they were still in the Gulf of Alaska in winter. In the homing adults, the pituitary contents and the plasma levels of gonadotropins and plasma sex steroid hormones peaked during upstream migration from the coast to the natal hatchery. The present results thus indicate that the seasonal increase in the activity of the PG axis is an important endocrine event that is inseparable from initiation of spawning migration of chum salmon.

Changes in gene expression for GH/PRL/SL family hormones in the pituitaries of homing chum salmon during ocean migration through upstream migration

Gene expression for growth hormone (GH)/prolactin (PRL)/somatolactin (SL) family hormones in the pituitaries of homing chum salmon were examined, because gene expression for these hormones during ocean-migrating phases remains unclear. Fish were collected in the winter Gulf of Alaska, the summer Bering Sea and along homing pathway in the Ishikari River-Ishikari Bay water system in Hokkaido, Japan in autumn. The oceanic fish included maturing adults, which had developing gonads and left the Bering Sea for the natal river by the end of summer. The absolute amounts of GH, PRL and SL mRNAs in the pituitaries of the maturing adults in the summer Bering Sea were 5- to 20-fold those in the winter Gulf of Alaska. The amount of GH mRNA in the homing adults at the coastal seawater (SW) areas was smaller than that in the Bering fish, while the amount of PRL mRNA remained at the higher level until fish arrived at the Ishikari River. The gill Na(+),K(+)-ATPase activity in the coastal SW fish and the plasma Na(+) levels in the brackish water fish at the estuary were lowered to the levels that were comparable to those in the fresh water (FW) fish. In conclusion, gene expression for GH, PRL and SL was elevated in the pituitaries of chum salmon before initiation of homing behavior from the summer Bering Sea. Gene expression for GH is thereafter lowered coincidently with malfunction of SW adaptability in the breeding season, while gene expression for PRL is maintained high until forthcoming FW adaptation.

Duplicated Kiss1 receptor genes in zebrafish: distinct gene expression patterns, different ligand selectivity, and a novel nuclear isoform with transactivating activity

The kisspeptin (Kiss1) and Kiss1 receptor (Kiss1r) pathway plays a central role in the neuroendocrine control of reproduction. In contrast to humans and mammals that have a single Kiss1 gene and a single Kiss1r gene, multiple Kiss ligand and receptor genes are found in nonmammalian vertebrates. Their functional relationship, however, is poorly understood. Here, we report that the duplicated zebrafish kiss1r genes have evolved a distinct gene expression pattern, different ligand selectivity, and novel nuclear isoforms. While a single kiss1ra mRNA was detected exclusively in the brain, 5 kiss1rb transcripts were found in many peripheral tissues. Functional assays showed that kiss1ra encodes a receptor activated by both Kiss1 and Kiss2, while kiss1rb encodes a receptor that has a preference for Kiss1. The four alternatively spliced kiss1rb mRNAs encoded 4 truncated isoforms, denoted kiss1rb‐derived protein (KRBDP)1–4. When their subcellular localization was examined, KRBDP3 and KRBDP4 were found in the nucleus in cultured mammalian cells and in zebrafish embryos. One‐hybrid transcription activation assays revealed that KRBDP3, but not KRBDP4, possesses ligand‐independent trans‐activation activity. These findings highlight how the duplication of Kiss1r genes may facilitate their adaptation of specialized functions. The discovery of a nuclear Kiss1r isoform raises the possibility of novel function of Kiss1r in the nucleus.—Onuma, T. A., Duan, C. Duplicated Kiss1 receptor genes in zebrafish: distinct gene expression patterns, different ligand selectivity, and a novel nuclear isoform with transactivating activity. FASEB J. 26, 2941–2950 (2012). www.fasebj.org

Elevation of the plasma level of insulin-like growth factor-I with reproductive maturation prior to initiation of spawning migration of chum salmon.

When, where, and how oceanic chum salmon initiate spawning migration is unknown although gonadal development and elevation of the activity of the pituitary–gonadal axis (PG‐axis) are essential. Insulin‐like growth factor‐I (IGF‐I) is a somatotropic signal that interacts with the PG‐axis for gametogenesis. We thus examined the plasma level of IGF‐I in immature and maturing chum salmon in the Bering Sea and the Gulf of Alaska. The maturing adults which had maturing gonads left the Bering Sea for the natal river by the end of summer, because almost all fish were immature in September. The plasma level of IGF‐I and corresponding body size in the maturing adults were two‐ to threefold that of immature fish. The plasma IGF‐I level correlated positively with the pituitary contents of follicle‐stimulating hormone and the plasma levels of 11‐ketotestosterone and estradiol‐17β. Therefore, the plasma level of IGF‐I increased with elevation of the PG‐axis activity prior to the initiation of spawning migration from the Bering Sea. Circulatory IGF‐I from visceral organs may inform the status of body growth to the PG‐axis for gonadal development that is inseparable from decision of chum salmon whether to initiate homing behavior from the Bering Sea or not to initiate spawning migration by the coming spawning season.

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.