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.     

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.     

Effect of salmon gonadotropic hormone on sex steroids in male rainbow trout: plasma levels and testicular secretion in vitro

Summary Male rainbow trout were treated with salmon gonadotropic hormone (GTH) at different stages of the circannual reproductive cycle; spawning fish were also treated with an antiserum against salmon GTH. Injection of GTH led to a several-fold increase of plasma sex steroid levels during spermatogenesis and in the spawning season but was without effect at early stages of testicular development. GTH neutralization during the spawning season was followed by a several-fold decrease of plasma sex steroid levels. During spermatogenesis and in the spawning season, both treatment regimes resulted in an increased sensitivity of testicular explants in response to a subsequent stimulation of steroid secretion in vitro. This up-regulatory response may facilitate and maintain the high sex steroid plasma levels observed during the spawning season. It may also be necessary to allow for concomitant peak values of plasma GTH and sex steroids in the spawning season, a situation difficult to understand within the negative feedback concept. The adaptive capacities of the testicular steroidogenic system indicate that it is not only an effector site for GTH but also an active part of the endocrine system controling reproduction.Abbreviations BSA bovine serum albumin - bw body weight - E₂ 17-estradiol - GnRH gonadotropin releasing-hormone - GTH gonadotropic hormone - LH luteinizing hormone - OHT 11-hydroxytestosterone - OT 11-ketotestosterone - 17-20P 17-hydroxy, 20-dihydroprogesterone - PE pituitary extract - raGTH rabbit anti-GTH antiserum - rPS rabbit preimmune serum - T testosterone     

Steroid and thyroid hormone profiles following a single injection of partly purified salmon gonadotropin or GnRH analogues in male and female sea lamprey

The effects of exogenous administration of gonadotropin-releasing hormone (GnRH) analogues or of a partly purified salmon gonadotropin extract (GTH) on the duration of steroid and thyroid hormone levels were determined in female and male sea lampreys, Petromyzon marinus, tested under differing temperature and reproductive status. Plasma estradiol levels, but not androgens, were significantly elevated in response to the GnRH analogues or GTH injection compared to controls in female and male lampreys. Higher temperature and/or advance in time of maturation appeared to be inversely related to plasma estradiol levels. These data provide further evidence of hypothalamic control over reproductive function in lampreys. Plasma thyroxine was significantly elevated after female lampreys were treated with GTH, GnRHa (10 micrograms/lamprey) or GnRHa (1 microgram/lamprey) compared to controls. The present study is the first to demonstrate that the GnRH analogue stimulated in some way the pituitary-thyroid axis. These data suggest that a GnRH activity may activate both gonado- and thyrotropic secretion or that the endogenous hormone may itself have both functions in one of the most primitive vertebrates, the sea lamprey.     

Homologous desensitization of gonadotropin-releasing hormone (GnRH) receptors in the goldfish pituitary: effects of native GnRH peptides and a synthetic GnRH antagonist.

Gonadotropin-releasing hormone (GnRH) stimulates release of gonadotropin hormone (GTH) through interaction with high affinity receptors in the goldfish pituitary. In the present study, we investigated desensitization of two native GnRH peptides, [Trp7, Leu8]-GnRH (sGnRH) and [His5, Trp7, Tyr8]-GnRH (cGnRH-II), using superfused fragments of goldfish pituitary in vitro. Pulsatile treatment with either sGnRH or cGnRH-II (2-min pulses given every 60 min) resulted in dose-dependent secretion of GTH from the goldfish pituitary; cGnRH-II had a greater GTH release potency and displayed a greater receptor binding affinity than sGnRH. Both sGnRH and cGnRH-II-induced GTH release were partially inhibited by concomitant treatment with either [D-Phe2, Pro3, D-Phe6]-GnRH or [D-pGlu1, D-Phe2, D-Trp3.6]-GnRH. These antagonists had greater receptor binding affinities than the native peptides, with no stimulatory action on GTH release in the absence of the GnRH agonists. Continuous treatment with either sGnRH or cGnRH-II (10(-7) M), rapidly desensitized pituitary GTH release in a biphasic fashion; initially there was a rapid increase in GTH release of approximately 10-20-fold (phase 1), followed by a sharp decline in GTH release, reaching a stable concentration 2-3-fold above the basal level (phase 2). Further stimulation of the pituitaries with sGnRH or cGnRH-II (10(-7) M) (second treatment) after 60 min recovery resulted in a significantly lower sGnRH or cGnRH-II-induced GTH release compared to that observed during the initial treatment period.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Inhibition of pituitary-gonadal function in male rats by a potent GnRH antagonist

The inhibitory effects of the potent GnRH antagonist, [Ac-D-pCl-Phe1,2,D-Trp3,D-Arg6,DAla10]GnRH (GnRHant) upon pituitary-gonadal function were investigated in normal and castrated male rats. The antagonist was given a single subcutaneous (s.c.) injections of 1-500 micrograms to 40-60 day old rats which were killed from 1 to 7 days later for assay of pituitary GnRH receptors, gonadal receptors for LH, FSH, and PRL, and plasma gonadotropins, PRL, and testosterone (T). In intact rats treated with low doses of the antagonist (1, 5 or 10 micrograms), available pituitary GnRH receptors were reduced to 40, 30 and 15% of the control values, respectively, with no change in serum gonadotropin, PRL, and T levels. Higher antagonist doses (50, 100 or 500 micrograms) caused more marked decreases in free GnRH receptors, to 8, 4 and 1% of the control values, which were accompanied by dose-related reductions in serum LH and T concentrations. After the highest dose of GnRHant (500 micrograms), serum LH and T levels were completely suppressed at 24 h, and serum levels of the GnRH antagonist were detectable for up to 3 days by radioimmunoassay. The 500 micrograms dose of GnRHant also reduced testicular LH and PRL receptors by 30 and 50% respectively, at 24 h; by 72 h, PRL receptors and LH receptors were still slightly below control values. In castrate rats, treatment with GnRHant reduced pituitary GnRH receptors by 90% and suppressed serum LH and FSH to hypophysectomized levels. Such responses in castrate animals were observed following injection of relatively low doses of GnRHant (100 micrograms), after which the antagonist was detectable in serum for up to 24 h. These data suggest that extensive or complete occupancy of the pituitary receptor population by a GnRH antagonist is necessary to reduce plasma gonadotropin and testosterone levels in intact rats. In castrate animals, partial occupancy of the available GnRH receptor sites appears to be sufficient to inhibit the elevated rate of gonadotropin secretion.     

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.     

Molecular cloning and sequencing of salmon gonadotropin beta subunit

Gonadotropin (GTH) was purified from the pituitaries of the Pacific chinook salmon using a combination of stepwise ethanol precipitation and concanavalin-A affinity chromatography. The alpha and beta subunits were dissociated and fractionated by C-18 reverse-phase high-performance liquid chromatography with a 0.1% trifluoroacetic acid/acetonitrile gradient. An enriched cDNA library was screened for the beta-GTH gene(s) using two synthetic oligonucleotides based on partial protein data. A positive, full-size clone (E3) was identified and sequenced. It contains 657 base pairs and codes for a 142-amino-acid precursor protein. The mature salmon beta-GTH (119 amino acids) is structurally homologous to human luteinizing hormone and chorionic gonadotropin. The effect of testosterone implantation on pituitary GTH and beta-GTH mRNA was examined with radioimmunoassay and Northern blot analysis. There was a corresponding increase in both the pituitary GTH and mRNA levels.     

Cloning and characterization of an odorant receptor in five Pacific salmon

The olfactory system of fish is extremely important as it is able to recognize and distinguish a vast of odorous molecules involved in wide ranges of behaviors including reproduction, homing, kin recognition, feeding and predator avoidance; all of which are paramount for their survival. We cloned and characterized one type olfactory receptors (ORs) from five congeneric salmonids: lacustrine sockeye salmon (Oncorhynchus nerka), pink salmon (O. gorbuscha), chum salmon (O. keta), masu salmon (O. masou) and rainbow trout (O. mykiss). Lacustrine sockeye salmon olfactory receptor 1 (LSSOR1) showed high sequence homology to the OR subfamily, and was expressed only in the olfactory epithelium (as indicated by PCR amplified genomic DNA and cDNA). OR genes from the five salmonids examined all showed strong homology (96-99%) to each other. Hypervariable regions, believed to be ligand-binding pockets, showed homologous completely matched amino acid sequences except for one amino acid in pink salmon olfactory receptor 1 (PSOR1), revealing that these ORs may be well conserved among salmon species. These results suggest that the isolated 5 salmonid ORs might play an important role in salmon life cycles.     

Comparative anatomy of the dorsal hump in mature Pacific salmon

Mature male Pacific salmon (Genus Oncorhynchus ) demonstrate prominent morphological changes, such as the development of a dorsal hump. The degree of dorsal hump formation depends on the species in Pacific salmon. It is generally accepted that mature males of sockeye (O. nerka ) and pink (O. gorbuscha ) salmon develop most pronounced dorsal humps. The internal structure of the dorsal hump in pink salmon has been confirmed in detail. In this study, the dorsal hump morphologies were analyzed in four Pacific salmon species inhabiting Japan, masu (O. masou ), sockeye, chum (O. keta ), and pink salmon. The internal structure of the dorsal humps also depended on the species; sockeye and pink salmon showed conspicuous development of connective tissue and growth of bone tissues in the dorsal tissues. Masu and chum salmon exhibited less‐pronounced increases in connective tissues and bone growth. Hyaluronic acid was clearly detected in dorsal hump connective tissue by histochemistry, except for in masu salmon. The lipid content in dorsal hump connective tissue was richer in masu and chum salmon than in sockeye and pink salmon. These results revealed that the patterns of dorsal hump formation differed among species, and especially sockeye and pink salmon develop pronounced dorsal humps through both increases in the amount of connective tissue and the growth of bone tissues. In contrast, masu and chum salmon develop their dorsal humps by the growth of bone tissues, rather than the development of connective tissue.     

Comparative response of rams and bulls to long-term treatment with gonadotropin-releasing hormone analogs

The objective was to compare the relative response between rams and bulls in characteristics of LH, FSH and testosterone (T) secretion, during and after long-term treatment with GnRH analogs. Animals were treated with GnRH agonist, GnRH antagonist, or vehicle (Control) for 28 days. Serial blood samples were collected on day 21 of treatment, and at several intervals after treatment. Injections of natural sequence GnRH were used to evaluate the capacity of the pituitary to release gonadotropins during and after treatment. Treatment with GnRH agonist increased basal LH and T concentrations in both rams and bulls, with a greater relative increase in bulls. Endogenous LH pulses and LH release after administration of GnRH were suppressed during treatment with GnRH agonist. Treatment with GnRH antagonist decreased mean hormone concentrations, LH and T pulse frequency, and the release of LH and T after exogenous GnRH, with greater relative effects in bulls. Rams previously treated with antagonist had a greater release of LH after administration of GnRH compared with control rams, while rams previously treated with agonist showed a reduced LH response. Bulls previously treated with agonist had reduced FSH concentrations and LH pulse amplitudes compared with control bulls while bulls previously treated with antagonist had greater T concentrations and pulse frequency. The present study was the first direct comparison between domestic species of the response in males to treatment with GnRH analogs. The findings demonstrated that differences do occur between rams and bulls in LH, FSH and testosterone secretion during and after treatment. Also, the consequences of treatment with either GnRH analog can persist for a considerable time after discontinuation of treatment.     

Comparison of in vitro and in vivo effects of different species specific GnRH and their analogs

Mammalian, salmon and chicken gonadotropin-releasing hormones (mGnRH, sGnRH, cGnRH) and their analogs were synthesized and tested for their ability to stimulate in vitro LH and FSH release from cultured and superfused rat pituitary cells and also their in vivo effect were investigated on the artificial propagation of fishes. The LH and FSH releasing activity of sGnRH, cGnRH and their analogs were lower than the appropriate mammalian ones from cultured rat pituitary cells, but two of the cGnRH analogs showed increased LH and FSH secretory activity from superfused rat pituitary cells compared to the mGnRH. At the same time these two analogs are very potent to stimulate reproductive function of fishes and using these peptides we were able to fulfill the artificial propagation of fishes which could not be artificially propagated before.     

Effects of testosterone on gonadotropins, testes, and plasma 17alpha,20beta-dihydroxy-4-pregnene-3-one levels in postbreeding mature Atlantic salmon, Salmo salar, male parr.

Atlantic salmon, Salmo salar, mature male parr were implanted with testosterone (T) in small (T3) or large (T10) Silastic capsules in the breeding season or at its end in November or December, in order to find out whether the postbreeding decline in 17alpha, 20beta-dihydroxy-4-pregnene-3-one (17,20beta-P) and milt production is a consequence of declining T levels. In the first of three experiments, fish were sampled the following January and March, whereas in the second and in the third they were sampled in April. Pituitary and plasma concentrations of gonadotropic hormone (GTHs) I and II and plasma levels of T, 11-ketotestosterone (11-KT) and 17, 20beta-P were measured by radioimmunoassays, and the testes were examined histologically. Administration of T prolonged the period in which running milt was present, suppressed Sertoli cells, and prevented the postbreeding decline in testes weight in experiment two. The postbreeding decline in plasma 17,20beta-P levels was diminished by T10 in experiment one, and by both T3 and T10 in experiment two. The similar decline in 11-KT levels was not influenced by T treatment (only studied in experiment one). T treatment also prevented a decline in pituitary GTH II content (in experiments two and three) and in plasma GTH II levels (only studied in experiment three). However, pituitary GTH I content was not influenced (experiment two and three), whereas plasma GTH I levels (only studied in experiment three) were suppressed by T. To summarize, T treatment prevents postbreeding decline in 17,20beta-P levels, probably via a stimulation of GTH II secretion. J. Exp. Zool. 284:425-436, 1999.     

Peptides interact in gonadotrophin regulation

The regulation of luteinizing hormone (LH) activity is vital to normal reproductive functioning of the female. Although gonadotrophin-releasing hormone (GnRH) has a prominent role in the regulation of LH it is now believed that other peptides are also involved. Among these peptides is oxytocin. The addition of oxytocin to cultures of pituitary cells from female rats elicited a concentration-dependent secretion of LH. This secretion was enhanced in an oestrogenised environment and was inhibited by progesterone and testosterone. Oxytocin administered to female rats at pro-oestrus advanced the endogenous LH surge that occurs on the evening of pro-oestrus. Conversely oxytocin receptor antagonist suppressed the production of the LH surge in a dose-dependent manner, indicating that endogenous oxytocin is a crucial component of LH regulation. In the human female, oxytocin administered during the late follicular phase advanced the onset of the midcycle LH surge. Oxytocin added to rat pituitary cells in vitro induced LH synthesis. Furthermore rats administered oxytocin on pro-oestrus had higher LH pituitary content following development of the LH surge than did rats administered saline. Thus oxytocin promoted synthesis and replacement in the pituitary of LH released into the circulation. Incubation of pituitary pieces with oxytocin plus GnRH induced secretion of amounts of LH greater than the sum of the amounts released by oxytocin and GnRH separately. Additionally the increased LH levels observed in the peripheral circulation of pentobarbitone-anaesthetised rats administered GnRH were enhanced if the rats received oxytocin prior to the GnRH. Thus oxytocin synergised with GnRH in stimulating LH release. Addition of diBucAMP reduced the oxytocin-mediated augmentation and dideoxyadenosine enhanced the augmentation, suggesting that oxytocin worked most efficiently in a milieu low in cAMP activity. The use of a cell immunoblot assay revealed that individual cells responded differently to oxytocin and to GnRH and that the two peptides could act on the same cell. Perifusion studies performed on hemipituitaries demonstrated that a LH response could be determined by the presence of three peptides, oxytocin, neuropeptide Y and GnRH. Hence oxytocin is potentially involved also in multiple interactions during the process of LH regulation. LH regulation is therefore apparently the result of a community of peptides acting in a co-operative network.