Isolation and characterization of two coho salmon gonadotropins, GTH I and GTH II

Two gonadotropins, GTH I and GTH II, were isolated from pituitaries of spawning coho salmon (Oncorhynchus kisutch) using sequential extractions with ammonium acetate (pH 9.0) and 40% ethanol, precipitation with 80% ethanol, gel filtration chromatography (Sephadex G-100), anion-exchange chromatography (Mono-Q Sepharose), and gel filtration chromatography (Sephadex G-75). Coho salmon GTH I and GTH II stimulated steroidogenesis in vitro in a similar dose-dependent manner when incubated with either ovaries or testes of prepubertal coho salmon. An in vivo bioassay using coho salmon parr demonstrated that coho salmon GTH I and GTH II did not contain thyrotropic activity. Molecular weights were estimated by gel filtration chromatography to be 43,000 and 39,000 for GTH I and GTH II, respectively. Analysis of coho salmon GTH I and GTH II on reversed-phase high-performance liquid chromatography (rpHPLC) revealed that they consist of alpha and beta subunits with N-terminal amino acid residues of Tyr, Gly (alpha, beta of GTH I) and Tyr,Ser (alpha, beta of GTH II). Coho salmon GTH I-beta and GTH II-beta differed from each other in amino acid composition, N-terminal amino acids (Gly vs. Ser), and molecular weights in SDS-PAGE (19,000 vs. 20,000) and had a high degree of chemical similarity to chum salmon GTH I-beta and GTH II-beta, respectively. Specific rabbit antisera to the beta subunits of coho salmon GTH I and GTH II were generated. The observation of two GTHs with distinctly different chemical characteristics in coho salmon is similar to what has previously been found in chum salmon.     

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

Insulin-like growth factor-I (IGF-I) has been shown to be involved in pubertal activation of gonadotropin (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 alpha (GPalpha), follicle-stimulating hormone (FSH) beta, and luteinizing hormone (LH) beta 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 GPalpha, FSHbeta, and LHbeta 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.     

Thyrotropic activity of salmon pituitary glycoprotein hormones in the Hawaiian parrotfish thyroid in vitro

The thyrotropic activities of salmon pituitary extract, thyroid-stimulating hormone (TSH), gonadotropins (GTH), and glycoprotein fractions obtained during purification of salmon TSH and GTH were measured using the parrotfish thyroid culture system. Purified salmon TSH was approximately 1,000 times more potent than bovine TSH in stimulating thyroxine release into the culture medium. Most of the forms of salmon GTH had no thyrotropic activity. One of the forms of salmon GTH (GTH-F) and three chromatofocusing fractions (CF-B, -C, and -E) that were devoid of activity in the coho salmon in vivo had some thyrotropic activity in the parrotfish thyroid culture. Whether the activity of these fractions was due to contamination with TSH, less potent forms of TSH, or inherent thyrotropic activity of a form of GTH is discussed. These results indicate that the parrotfish thyroid culture system can be used to detect thyrotropic activity of fractions obtained during the purification of teleost TSH.     

Effects of a gonadotropin-releasing hormone antagonist on gonadotropin levels in Masu salmon and Sockeye salmon

The salmon gonadotropin-releasing hormone (sGnRH) is considered to be involved in gonadal maturation via gonadotropin (GTH) secretion in salmonid fishes. However, there is no direct evidence for endogenous sGnRH-stimulated GTH secretion in salmonids. In this study, to clarify whether endogenous sGnRH stimulates GTH secretion, we examined the effects of the mammalian GnRH (mGnRH) antagonist [Ac-Delta(3)-Pro(1), 4FD-Phe(2), D-Trp(3,6)]-mGnRH on luteinizing hormone (LH) levels in 0-year-old masu salmon Oncorhynchus masou and sockeye salmon Oncorhynchus nerka. First, the effects of the GnRH antagonist on LH release were examined in 0-year-old precocious male masu salmon. GnRH antagonist treatment for 3 hr significantly inhibited an increase in plasma LH levels that was artificially induced by exogenous sGnRH administration, indicating that the GnRH antagonist is effective in inhibiting LH release from the pituitary. Subsequently, we examined the effect of the GnRH antagonist on LH synthesis in 0-year-old immature sockeye salmon that were pretreated with exogenous testosterone for 42 days to increase the pituitary LH contents; the testosterone treatment did not affect the plasma LH levels. GnRH antagonist treatment slightly but significantly inhibited an increase in the testosterone-stimulated pituitary LH content levels. However, no significant differences in the plasma LH levels were observed between the GnRH antagonist-treated and control groups. These results suggest that endogenous sGnRH is involved in LH secretion in salmonid fishes.     

Cloning and sequencing of the cDNA encoding the pituitary gonadotropin II β-subunit of yellowfin porgy (Acanthopagrus latus)

The complementary DNA (cDNA) encoding the gonadotropic hormone (GTH) II pre-β-subunit of yellowfin porgy ( Acanthopagrus latus ) was isolated from a pituitary gland cDNA phage library. The cDNA insert, 598 base pairs (bp), contained a 411 bp open reading frame with 35 bp and 152 bp flanking regions at the 5'- and 3'-ends, respectively. The deduced amino acid sequence revealed a putative signal peptide of 24 amino acid residues and a 113 amino acid mature β-subunit of GTH polypeptide. This pre-β-subunit polypeptide of the yellowfin porgy GTH II showed 86% sequence identity with that of bonito GTH II-β 73% with killifish GTH II-β, 63% with African catfish GTH II-β 61% with pike eel GTH-β, 60% with silver carp GTH-β, 59% with chum salmon GTH 11-β, 58% with common carp GTH-β 57% with chinook Pacific salmon GTH-β, and 53% with European eel GTH II-β.     

Effects of photoperiod on pituitary gonadotropin levels in masu salmon

We examined the effects of photoperiod on pituitary levels of two types of gonadotropin (GTH), GTH I and GTH II, in masu salmon Oncorhynchus masou to study their mechanism of synthesis. In Experiment 1, the effects of long or short photoperiod combined with castration were examined using 8-month-old precocious males. Castration was carried out in early August and then the fish were reared under a short (8L16D) or long (16L8D) photoperiod for 60 days. In Experiment 2, the effects of photoperiod combined with testosterone treatment were examined using 12-month-old immature females. Silastic tubes containing testosterone (500 microg /fish) or vehicle were implanted intra-peritoneally in early October. Fish were reared under 16L8D for 60 days, and then half of the fish were transferred to 8L16D, while the remaining fish were kept under 16L8D until Day 90. In Experiment 1, GTH I contents were higher under 16L8D than under 8L16D in the castrated group on Day 30. Moreover, GTH I contents were higher in the castrated group than the control group under 16L8D on Day 30. GTH II contents increased with testicular maturation in the control groups, whereas they remained at low levels in the castrated groups regardless of photoperiodic treatment. In Experiment 2, GTH I contents did not change remarkably in all the groups, while GTH II contents were remarkably increased by testosterone treatment regardless of photoperiodic treatment. These results indicate that the synthesis of GTH I and GTH II are differently regulated by photoperiod and testosterone in masu salmon.     

Molecular cloning and characterization of complementary DNA encoding for the gonadotropin α-subunit of yellowfin porgy (Acanthopagrus latus)

Recombinant phage clones containing the complementary DNA (cDNA) coding for the a-subunit of gonadotropin (GTH) were isolated from a pituitary gland cDNA library of yellowfin porgy ( Acanthopagrus latus ) by using a degenerate oligonucleotide based on the conserved amino acids sequence of GTH from other species as a probe. This section of 735 base pairs (bp) cDNA contained a 351 bp open reading frame with 20 bp and 345 bp flanking regions at the 5'- and 3'-ends, respectively. The deduced amino acid sequence revealed a 23 amino acid signal peptide and a 94 amino acid mature α-subunit of GTH polypeptide. The latter polypeptide showed 66, 62 and 61% amino acid identity with chum salmon, pike eel and carp GTH peptide hormone α-subunit, respectively.     

Development of an enzyme-linked immunosorbent assay for goldfish gonadotropin

An enzyme-linked immunosorbent assay (ELISA) for goldfish gonadotropin (GTH) was developed with the intent of devising a simple, reliable and nonradioisotopic assay for the measurement of GTH in goldfish biological samples. In this assay, soluble GTH of the standards or samples competes with carp GTH (cGTH) immobilized on a solid support (96-well microplate) for the fixation on antibodies to the beta-subunit of carp gonadotropin. The immobilized antigen-antibody complexes are then revealed by the peroxidase-antiperoxidase (PAP) technique. After revelation of the peroxidase activity, the absorbance value of each well is measured with a microplate reader. The cGTH concentration used for coating the wells is 2 ng/ml and the final dilution of the specific antibody is 1:80,000. The assay can be performed within 24 h and can be used over a range of 0.125-4 ng/ml. At about 50% binding, the intra- and interassay coefficients of variation are 5% and 9% respectively. The displacement curves generated by goldfish plasma or pituitary perifusion fractions were strictly parallel to the standard cGTH. In addition, the stimulation by salmon gonadotropin-releasing hormone of pituitary fractions perifused in vitro caused an immediate increase in the GTH measured in the collected fractions, strongly reinforcing the assumption that this assay indeed measures GTH.     

Regulation of gonadotropin beta subunit gene expression by testosterone and gonadotropin-releasing hormones in the goldfish, Carassius auratus

Sex steroids differentially regulate gonadotropin (GTH) beta subunits (FSHbeta and LHbeta) gene expression in the pituitary of goldfish: a strong in vivo inhibitory effect on FSHbeta mRNA production, but a weak stimulatory effect on LHbeta in sexually immature and recrudescent fish. In the present study, to examine a direct effect of testosterone (T) and gonadotropin-releasing hormone (GnRH) on the mRNA levels of FSHbeta and LHbeta subunits in the pituitary, in vitro experiments were performed using dispersed pituitary cells of sexually immature, recrudescent, mature and regressed goldfish. T treatment in vitro did not significantly decrease FSHbeta mRNA levels, but increased that of LHbeta only in the cells of immature fish. Salmon-type GnRH increased FSHbeta mRNA levels in cells of mature fish, but decreased the levels in cells of sexually regressed fish. From these results, it was suggested that: (1) in vivo effect of sex steroids on gene expression of GTH beta subunits is not always exerted on the pituitary; and (2) the different responses of GTH beta subunits by sex steroids between in vivo and in vitro are partly due to a complex pathway through hypothalamic factors, such as GnRH, in the case of in vivo.     

The involvement of gonadotrophin and sex steroids in the control of reproduction in the parr and adults of Atlantic salmon, Salmo salar L.

Early sexual maturity occurred in the majority of male Atlantic salmon parr. Levels of the plasma androgens testosterone and 11-ketotestosterone rose steadily as the male parr matured, and decreased as the testes regressed. No such progressive changes were observed in the plasma gonadotrophin (GTH) levels, although the pituitary GTH levels were much higher in mature than in immature male parr; reasons for this, incluiding the possibility that the GTH radioimmunoassay employed is inadequate, are discussed. All female parr remained immature throughout the year, although the gonadosomatic index did show an annual cycle. Adult salmon had higher GTH and sex steroid levels than parr at the same stage of sexual maturity, the levels corresponding to the degree of sexual development of the adult fish.     

A two-receptor model for salmon gonadotropins (GTH I and GTH II).

The possible existence of distinct receptors for salmon gonadotropins (GTH I and GTH II) and the distribution of the receptor(s) were studied through examination of the binding of coho salmon (Oncorhynchus kistuch) GTH I and GTH II to membranes from thecal layers and granulosa cells of salmon ovaries. Purified coho salmon gonadotropins were iodinated by the lactoperoxidase method. Crude membrane preparations were obtained from thecal layers, granulosa cells, and whole ovaries of coho salmon in the postvitellogenic/preovulatory phase. Binding of 125I-GTH I to membranes from thecal layers, granulosa cells, and whole ovaries, and binding of 125I-GTH II to thecal layer cell membranes could be inhibited by both GTHs, but GTH I was more potent than GTH II. In contrast, GTH II was more potent than GTH I in inhibiting 125I-GTH II binding to membranes from granulosa cells and whole ovaries, but the inhibition curves were not parallel. Scatchard plot analysis suggested that there was a single type of receptor in the thecal layers for both GTHs, whereas in the granulosa cells there was more than one type of receptor for both GTHs. Based on these results, a two-receptor model for the postvitellogenic/preovulatory salmon ovary is proposed with the following features: 1) there are two types of gonadotropin receptors in the salmon ovary, type I and type II; 2) the type I receptor binds both GTHs, but with higher affinity for GTH I, whereas the type II receptor is highly specific for GTH II and may have only limited interaction with GTH I; and 3) the type I receptor is present in both thecal cells and granulosa cells, whereas the type II receptor is present in granulosa cells.     

Immunoreactivity to gonadotropin-releasing hormone and gonadotropic hormone in the brain and pituitary of the rainbow trout Salmo gairdneri

Summary Immunoreactivity to gonadotropin-releasing hormone (GnRH) and gonadotropic hormone (GTH) was studied at the light-microscopical level in the brain and pituitary of rainbow trout at different stages of the first reproductive cycle using antisera against synthetic mammalian GnRH and salmon GTH. GnRH perikarya were localized exclusively in the preoptic nucleus, both in the pars parvicellularis and the pars magnocellularis. A few somata contacted the cerebrospinal fluid. Not all neurosecretory cells were GnRH-positive, indicating at least a bifunctionality of the preoptic nucleus. We recorded no differences between sexes or stages of gonadal development in the location of GnRH perikarya, whereas gradual changes were found in staining intensity during the reproductive cycle. GnRH fibres ran from the partes parvicellularis and magnocellularis through the hypothalamus and merged into a common tract at the transverse commissure before entering the pituitary. In the pituitary, GnRH was localized in the neural tissue of the neurointermediate lobe and, to a lesser extent, in the neural protrusions penetrating the proximal pars distalis. The bulk of GTH-positive cells was situated in the proximal pars distalis. Some cells were found more rostrally amidst prolactin cells or in the neurointermediate lobe. Only a limited number of GTH cells appeared to be in close contact with GnRH-positive material.     

大鳞大马哈鱼垂体促甲状腺素和促性腺激素分离纯化的研究

本文报道应用ConA-Sepharose 4B.亲和层析、凝胶过滤层析、离子交换层析及免疫亲和层析等技术,首次从大鳞大马哈鱼垂体中分离纯化了促甲状腺素(sTSH)和促性腺激素(sGTH)。在TSH生物测定中,纯化的。sTSH制品具有明显促进虹鳟幼鱼甲状腺体内分泌甲状腺素(T4)的生物活性,而sGTH无此活性。在GTH生物测定中,制备的sGTH具有显著诱导虹鳟卵母细胞体外培养成熟(GVBD)及分泌17α、20β-二氢孕酮的能力,而sTSH无此活性。HPLC表明sTSH和sGTH的分子量分别为27500和38000道尔顿。作者用SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)和等电聚焦检测了激素的纯度及等电点,用RIA或ELISA方法测定了几种垂体激素在sTSH终产物中的污染程度。     

Immunocytochemical studies of the gonadotropic cells in the pituitary gland of male mullet, Mugil cephalus, during the annual reproductive cycle in both natural habitat and captivity

Using antiserum specific for the β subunit of coho salmon gonadotropic hormone II (GTH II), an immunocytochemical study of Mugil cephalus (L.) pituitaries was conducted during the annual reproductive cycle of the male in both natural habitat and captivity. The gonadotropic potency of the pituitary gland in general underwent an obvious increase during testicular development, reaching a peak at the time of reproductive maturity. During the testicular cycle of M. cephalus, the GTH cells showed an increase in immunoreactive staining intensity, granulation, hypertrophy and hyperplasia during sexual maturation. However, degranulation, vacuolization, and weakened immunoreactivity of these cells occurred during spawning. The GTH cells in the pituitary gland of M. cephalus males reared in captivity appeared with high synthetic and secretory activity but the reproductive activity declined, as reflected in the form of low values of the gonadosomatic index (GSI) and earlier resorption of the testes.     

In vitro induction of luteinizing hormone synthesis by estrogen in organ-cultured pituitary glands of the Japanese eel,Anguilla japonica

An organ culture method for pituitary glands isolated from immature Japanese eels (Anguilla japonica) was developed. This method could conserve the histological features of the pituitary glands for at least 21 days. The ability to synthesize gonadotropic hormone (GTH) in cultured eel pituitary glands was examined by detecting luteinizing hormone (LH) beta protein immunohistochemically. In a basal medium (Leibovitz L-15), LH beta-immunoreactive cells were very scarce, but after addition of estradiol-17beta (E2) a large number of immunoreactive cells appeared, particularly in the proximal pars distalis. The stimulatory effects of E2 on LH beta synthesis were dose (1-100 ng/ml)- and time (1.5-7 days)-dependent. Thus, in contrast with previous reports of the lack of a direct effect of E2 on GTH synthesis in primary cultured eel pituitary cells, the present results clearly indicate that E2 can stimulate GTH synthesis in immature eel pituitary glands. This organ culture method is useful to examine the actions of steroids and also other endocrine factors on the eel pituitary gland.     

Functional relationship between receptor binding and biological activity for analogs of mammalian and salmon gonadotropin-releasing hormones in the pituitary of goldfish (Carassius auratus)

The relationship between gonadotropin-releasing hormone (GnRH) receptor binding and biological activity in the goldfish pituitary for mammalian and salmon GnRH (sGnRH) analogs with structural modification at the C terminus involving replacement of glycine amide with an alkyl amine and replacement of the Gly6 residue with D amino acids was examined. The GnRH receptor binding data were analyzed with a computerized curve-fitting program (LIGAND) for a single as well as two classes of binding sites; analysis based on one site fit estimated binding affinity and capacity for one class of binding site, and analysis based on two-site fit estimated binding affinity and capacity for two classes of binding sites (high-affinity/low-capacity and low-affinity/high-capacity binding sites). The estimated receptor affinity values were then used to determine the correlation between binding affinity and gonadotropin (GTH)-release potency in vitro. The highest correlation between biological activity and receptor binding affinity was obtained for the high-affinity/low-capacity binding sites and GnRH analogs containing Trp7 and Leu8 residues (i.e., the salmon GnRH structural format) (R = 0.940 +/- 0.150). For the same group of GnRH analogs, there was no significant correlation between the relative GTH-release potency and binding affinity of the low-affinity/high-capacity sites (R = 0.159 +/- 0.434), or that obtained from a one-site fit (R = 0.198 +/- 0.431). Similarly, for mammalian GnRH analogs, significant correlation between binding affinity and biological activity (R = 0.406 +/- 0.049) was only obtained for the high-affinity sites, although the degree of correlation was significantly lower than that obtained for salmon GnRH analogs. The present findings provide strong support for the hypothesis that high-affinity GnRH receptors are involved in the control of GTH release in the goldfish pituitary. In addition, the results demonstrate clearly that the presence of Trp7, Leu8 residues in salmon GnRH molecule, a native peptide in goldfish, is important for recognition of the ligand by the GnRH receptors in the goldfish pituitary, and that structural modifications at positions 6 and 10 in this peptide can increase receptor binding affinity and biological activity at the pituitary level. The most active sGnRH analog identified to date is [D-Arg6, Pro9-NEt]-sGnRH.     

Cloning, functional characterization, and expression of a gonadotropin receptor cDNA in the ovary and testis of amago salmon (Oncorhynchus rhodurus).

A gonadotropin receptor was cloned from amago salmon (Oncorhynchus rhodurus) ovarian follicles. This receptor (sGTH-R) belongs to the glycoprotein hormone receptor family with a large extracellular and seven-transmembrane domains. Its sequence homology is highest with mammalian LH receptors. Phylogenetic analysis reveals that sGTH-R is grouped with mammalian and chicken FSH and LH receptors, but not with mammalian TSH receptors. sGTH-R is expressed dominantly in the ovary and testis. Functional characterization examined with transiently transfected mammalian cells revealed increased intracellular cAMP level when exposed to mammalian and fish gonadotropins; the most potent hormone was salmon GTH II. These results indicate that the cloned cDNA encodes a functional amago salmon GTH receptor protein.