Masafumi Amano

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.

Changes in levels of GnRH in the brain and pituitary and GTH in the pituitary in male masu salmon, Oncorhynchus masou, from hatching to maturation.

Levels of two types of gonadotropin-releasing hormone (salmon GnRH and chicken GnRH-II) in the brain and pituitary, and content of gonadotropin (GTHIβ and IIβ) in the pituitary were measured in male masu salmon from hatching to gonadal maturation for three years in order to clarify the involvement of GnRHs in precocious maturation.Underyearling precocious males were distinguishable in summer of year 1 and were marked by an increased GSI. Spermiation was observed among these individuals thereafter every autumn. Pituitary GTHIβ content in both precocious and immature males, and GTHIIβ content in precocious males showed seasonal fluctuations — high in autumn and low in winter. Pituitary GTHIIβ content was low in immature males.Pituitary sGnRH content in precocious males increased from spring to autumn during the three-year period. sGnRH concentrations in discrete brain areas showed seasonal changes — high during autumn to winter and low in summer. Concentrations in the olfactory bulbs and hypothalamus increased significantly in association with testicular maturation during year 3. sGnRH concentrations in the hypothalamus were significantly higher in precocious males than in immature males; this was possibly due to positive feedback of steroid hormones. cGnRH-II was undetectable in the pituitary and no distinct changes were observed in its concentration in the brain in relation to maturation.The phenomenon of underyearling precocious maturation is considered to be triggered before the onset of early summer. It is suggested that males which mature precociously are larger in size and contain much sGnRH in the pituitary before the outward signs of precocity appear; sGnRH may stimulate GTH II synthesis and induce precocious maturation.RésuméLes contenus cérébraux et hypophysaires des deux types de gonadolibérines (GnRH de saumon et GnRH type II de poulet), et les contenus en gonadotropines hypophysaires (GTHIβ et GTHIIβ), ont été étudiés chez le saumon masu pendant 3 ans, de léclosion à la maturation des gonades pour tenter dexpliquer le rôle éventuel du sGnRH dans les processus de maturation précoce.Les mâles précoces sont détectés lété de la première année et caractérisés sur la base de laccroissement de leur rapport gonado-somatique (RGS). Chez ces animaux, la spermiation est ensuite observée tous les automnes. La charge hypophysaire en GTHIβ des mâles immatures et précoces, et en GTHIIβ des mâles précoces montre des variations saisonnières importantes en automne et faibles en hiver. Les contenus hypophysaires en GTHIIβ des mâles immatures restent faibles.Les contenus hypophysaires de sGnRH des mâles précoces augmentent chaque année entre le printemps et lautomne, durant les 3 ans de létude. Les contenus en sGnRH des aires cérébrales varient avec les saisons et sont importantes durant lautomne et lhiver et faibles en été. Les concentrations dans le bulbe olfactif et lhypotalamus augmentent significativement, corrélées avec lavancement de la spermatogénèse. Les concentrations de sGnRH de lhypothalamus des mâles précoces sont significativement plus élevées que celles des animaux immatures; sans doute liées à un feedback stéroïdien. Le cGnRH-II est indétectable dans lhypophyse et ses concentrations dans le cerveau ne varient pas avec le stade de maturité.Le phénomène de maturation précoce est supposé être initié avant le début de lété. II est supposé que les mâles précoces croissent plus vite et que leur hypophyse contient plus de sGnRH avant que les signes extérieurs de précocité se manifestent; ce sGnRH pourrait stimuler la synthèse de GTHII et induire en conséquence la maturation précoce.

Gonadotropin-releasing hormone and gonadotropin in goldfish and masu salmon

Reproductive activities in vertebrates are regulated by an endocrine system, consisting of the brain-pituitary-gonad axis. In teleosts, gonadotropin-releasing hormone (GnRH) in the brain stimulates gonadotropin (GTH) release in the pituitary gland, but because of lack of the portal vessel, it is not known when and how much GnRH is released for the regulation of GTH release. There are multiple molecular types of GnRH in teleosts and several distinct populations of GnRH neurons in the brain. However, we do not know which types and populations of GnRH neurons regulate reproductive activities. Here we summarize our recent studies on GnRH and GTH in masu salmon Oncorhynchus masou and goldfish Carassius auratus. Immunocytochemistry showed the location and molecular types of GnRH neurons. Salmon (sGnRH) and chicken-II GnRH (cGnRH-II) neuronal fibers were widely distributed in the brain of both masu salmon and goldfish. Only sGnRH fibers were observed in the pituitary of masu salmon, whereas both sGnRH and cGnRH-II fibers were observed in the goldfish pituitary, indicating that species specific GnRH profiles are involved in the regulation of pituitary function in teleosts. A series of experiments in masu salmon and goldfish suggest that among GnRH neuron populations GnRH neurons in the ventral telencephalon and the hypothalamus regulate GTH release, and that GnRH of the terminal nerve origin is not essential to gonadal maturation and ovulation. The biological function of other GnRH neurons remains unkown. Two GTHs appear to be characteristic of teleost; however, regulation of reproduction by these GTHs is a question that remains to be elucidated. In salmonid species, it is proposed that GTH I stimulates early gonadal development, whereas GTH II acts in later stages. When GTH expression was examined in goldfish, both GTH I β and II β mRNA levels in the pituitary gland showed increases in accordance with gonadal development, unlike the sequential expression of GTH subunits in salmonids. The expression of these GTH subunit mRNAs were affected by water temperature, starvation, and steroid hormones in goldfish, but in what manner these two GTHs regulate gonadal development remains to be clarified.

Chromatographic and immunological identification of gonadotropin-releasing hormone in five marine teleosts.

Brain extracts from bluefin tuna, Thunnus thynnus, red seabream, Pagrus major, black seabream, Acanthopagrus schlegeli, red spotted grouper, Epinephelus akaara and Japanese flounder, Paralichthys olivaceus, were analyzed by high performance liquid chromatography (HPLC) and specific radioimmunoassays. Immunoreactive material co-eluting from HPLC with salmon gonadotropin-releasing hormone (GnRH) and chicken GnRH-II, respectively, was found in all five species. In addition, a GnRH immunoreactive fraction showing the same HPLC retention time as lamprey GnRH-I was detected in the brain extracts of all species examined when using an unspecific radioimmunoassay which detects several GnRH forms, including lamprey GnRH-I. In the Japanese flounder brain extract, a fourth GnRH immunoreactive fraction was detected with the unspecific radioimmunoassay which did not co-elute with any of the six synthetic GnRH standards used in the present study.

The biased intracellular accumulation of the β-subunit of salmon gonadotropin (GTH II) in the pituitary of rainbow trout Oncorhynchus mykiss, during gametogenesis

Salmon gonadotropin (GTH II) is a heterodimeric glycoprotein hormone (α and IIβ subunits), serving as a maturational GTH, and is produced in a specific gonadotropic cell-type (GTH II-cells) containing small granules and large globules. In trout GTH II-cells, double immunolabeling for the α- and IIβ-subunits shows that colocalization of the α- and IIβ-immunolabeling is confined to the small granules, indicating storage of functional GTH II. On the other hand, α-immunolabeling is absent in the large globules, even though IIβ labeling is abundant throughout the period of seasonal gametogenesis. The α-specific antiserum recognizes the intact α-subunit as well as the reduced and deglycosylated α-subunits by immunoblotting. These results indicate that an accumulation of the IIβ-subunit is specifically generated in the large globules of these cells. In fact, with sexual maturity, the quantity of IIβ-subunits becomes elevated in the trout pituitary due to a marked increase in GTH II-cells containing many large globules. However, the derivation and function of the large globules and the fate of their contained IIβ-subunits remains unknown.

Effects of Olfactory Tract Section on the Immunohistochemical Distribution of Brain GnRH Fibers in the Female Goldfish, Carassius auratus

Abstract The organization of the gonadotropin-releasing hormone (GnRH) system originating from the terminal nerve (TN) neuron in the olfactory bulb was examined immunohistochemically by tracing the changes in the distribution of salmon type (sGnRH) and chicken-II type (cGnRH-II) GnRH fibers after olfactory tract section (OTX) in the female goldfish. Following OTX, which blocks the axonal transport of GnRH from the TN to other brain regions, the density of sGnRH-ir fibers in various brain regions decreased from 7 days to 28 days, with the exception of rather restricted areas surrounding cell bodies in the ventral telencephalon, preoptic area, and hypothalamus. The density of cGnRH-II-ir fibers decreased only in the telencephalon from 7 days to 14 days. In spite of the decrease of GnRH-ir fibers in several brain areas, neither type of GnRH fibers showed marked changes in the pituitary gland during the experiment. These results indicate that the TN GnRH system project fibers to wide brain areas and that most of sGnRH and some of the cGnRH-II-ir fibers in the brain of goldfish are TN origin. Furthermore, the GnRH system that project fibers to the pituitary does not primarily originate from the TN-GnRH system.