Accumulation of tetrodotoxin and 4,9-anhydrotetrodotoxin in cultured juvenile kusafugu Fugu niphobles by dietary administration of natural toxic komonfugu Fugu poecilonotus liver.

Non-toxic cultured juvenile kusafugu Fugu niphobles were fed with a diet containing highly toxic natural komonfugu Fugu poecilonotus liver until the 30th day (8.0 microg of TTX and 3.7 microg of 4,9-anhydroTTX/fish/day), and then fed with a non-toxic diet until the 240th day. During the 30-240th day, five or six fish were periodically sampled six times, and the contents of TTX and 4,9-anhydroTTX in each tissue were determined. The total TTX and 4,9-anhydroTTX accumulated in all tissues tested was not significantly changed during the experimental period, both being kept at 70% of administrated doses. However, in the liver, the TTX content accounted to be 120 microg (50% of administrated) on the 30th day, and then it gradually decreased to 50 microg until the 240th day, while 4,9-anhydroTTX content was kept at approximately 40 microg (40% of administrated) during all the experimental periods. In contrast to the liver, in the skin, TTX and 4,9-anhydroTTX were 40 and 5 microg, respectively, on the 30th day, and then gradually increased to 80 and 24 microg, respectively, until the 240th day. In the intestine, TTX and 4,9-anhydroTTX contents were kept at 25 and 12 microg, respectively, during all the experimental periods. According to these results, we assumed that a part of TTX accumulated in the liver was slowly transferred to the skin.

Examination of transformation among tetrodotoxin and its analogs in the living cultured juvenile puffer fish, kusafugu, Fugu niphobles by intramuscular administration.

In puffer fish, tetrodotoxin (TTX) exists as the major toxin with chemically equilibrium analogs (4-epiTTX, 4,9-anhydroTTX) and chemically non-equilibrium analogs (deoxy analogs, 11-oxoTTX, 4-S-cysteinylTTX). There are two purposes to this study: 1) to search for the reason why TTX is the most major analog in puffer fish, even 4,9-anhydroTTX is chemically more stable, 2) to investigate whether or not chemically non-equilibrium analogs are transformed in puffer fish, because these were predicted to be biosynthetic intermediates. Pure TTX, 4-epiTTX, 4,9-anhydroTTX, and 11-oxoTTX were separately administrated to the cultured non-toxic juvenile puffer fish kusafugu, Fugu niphobles by intramuscular injection. Sixteen days after administration, TTX analogs in the whole fish were analyzed by LC-fluorescent detection and LC/MS. By the administration of TTX, 4-epiTTX, and 4,9-anhydroTTX, 34-40% of the administrated doses of the toxins were accumulated, and 4,9-anhydroTTX has become the major toxin after inter-conversion. This result indicates discrepancy from the previous ones wherein TTX was predominantly accumulated when TTXs were administrated through diets; this suggests that dietary administration might be necessary to accumulate TTX as the major toxin, and not 4,9-anhydroTTX. Transformations from TTX to deoxy analogs or 11-oxoTTX, or from 11-oxoTTX to TTX were not detected in this study.

Changes in brain seabream GnRH mRNA and pituitary seabream GnRH peptide levels during ovarian maturation in female barfin flounder.

The pleuronectid barfin flounder Verasper moseri expresses three forms of gonadotropin-releasing hormones (GnRHs), i.e., seabream GnRH (sbGnRH), salmon GnRH, and chicken GnRH-II. Among these, sbGnRH is the dominant form in the pituitary, indicating that sbGnRH regulates gonadal maturation. In order to clarify the physiological roles of sbGnRH during ovarian maturation in reared female barfin flounder, the changes in brain sbGnRH mRNA levels and pituitary sbGnRH peptide levels were examined by real-time quantitative PCR and time-resolved fluoroimmunoassay, respectively. The fish hatched in April 2002. The gonadosomatic index remained low until August 2004 and increased thereafter until April 2005 when the fish began to ovulate. The sbGnRH mRNA levels per brain increased significantly from April 2004 to April 2005. Pituitary sbGnRH peptide levels also increased significantly during this period. These results indicate that sbGnRH is involved in ovarian maturation and ovulation in the barfin flounder.

Changes in the immunostaining intensities of follicle-stimulating hormone and luteinizing hormone during ovarian maturation in the female Japanese flounder.

The role of gonadotropin (GTH) in the reproduction of the Japanese flounder, Paralichthys olivaceus, was studied by assessing the changes in the apparent activity of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the pituitary gland during gonadal maturation by immunohistochemical analyses. Corresponding changes in plasma levels of testosterone (T), estradiol-17beta (E(2)), and 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP) were also studied. Reared fish at the early spawning to termination stages were sampled from May to August and wild fish at the previtellogenic to termination stages were caught at 3- to 4-week intervals between April and September offshore from the northern mainland of Japan by gill nets. The gonadosomatic index of the reared fish decreased from the early spawning stage to the termination stage, while that of the wild fish increased significantly from the previtellogenic stage to the early spawning stage and decreased thereafter. In the reared fish, the immunostaining intensities of FSH and LH were high during the spawning period, accompanied by high plasma levels of T, E(2), and DHP. In the wild fish, the immunostaining intensities of FSH and LH were low during the previtellogenic stage but increased during the maturing and spawning stages. These results indicate that both FSH and LH are likely associated with oocyte maturation in the Japanese flounder.

RFamide peptides inhibit the expression of melanotropin and growth hormone genes in the pituitary of an Agnathan, the sea lamprey, Petromyzon marinus.

Neuropeptides with the Arg-Phe-amide motif at their C termini (RFamide peptides) were identified in the brains of several vertebrates, and shown to have important physiological roles in neuroendocrine, behavioral, sensory, and autonomic functions. The present study identified RFamide peptides, which are teleost prolactin-releasing peptide (PrRP) homologs, in the sea lamprey, Petromyzon marinus and characterized their effect on the release of pituitary hormones in vitro. Two RFamide peptides (RFa-A and RFa-B) were isolated from an acid extract of sea lamprey brain, including hypothalamus by Sep-Pak C18 cartridge, affinity chromatography using anti-salmon PrRP serum, and reverse-phase HPLC on an ODS-120T column. Amino acid (aa) sequences and mass spectrometric analyses revealed that RFa-A and RFa-B consist of 25 and 20 aa, respectively, and have 75% sequence identity within the C-terminal 20 aa. The RFa-B cDNA encoding a preprohormone of 142 aa was cloned from the lamprey brain, and the deduced aa sequence from positions 48-67 was identical to the sequence of RFa-B. However, the preprohormone does not include an aa sequence similar to the RFa-A sequence. Cell bodies, which were immunoreactive to anti-salmon PrRP serum, were located in the periventricular arcuate nucleus, ventral part of the hypothalamus, and immunoreactive fibers were abundant from the hypothalamus to the brain. A small number of immunoreactive fibers were detected in the dorsal half of the rostral pars distalis of the pituitary, close to the GH-producing cells. In addition, anti-salmon PrRP immunoreactivities were observed in the pars intermedia, corresponding to melanotropin cells. Likewise, signal of RFa-B mRNA was detected not only in the brain but also in the pars intermedia. The synthetic RFa-A and -B inhibited GH mRNA expression in a dose-dependent fashion in vitro, which is comparable to the inhibitory effect of teleost PrRP on GH release. Both RFa-A and -B also inhibited the expression of proopiomelanotropin mRNA, but no effects were observed in the expression of proopiocortin and gonadotropin beta mRNAs. The results indicate that RFamide peptides, which are teleost PrRP homologs, are present in the hypothalamus and pituitary of sea lamprey, and may be physiologically involved in the inhibition of GH and melanotropin release in the sea lamprey pituitary.

Immunohistochemical localization and ontogenic development of prolactin-releasing peptide in the brain of the ovoviviparous fish species Poecilia reticulata (guppy).

Immunohistochemical localization and ontogenic development of prolactin-releasing peptide (PrRP) in the brain of the ovoviviparous fish species Poecilia reticulata (guppy) were examined to gain a better understanding of this hormone in teleost fish. In adult guppies, PrRP-immunoreactive (ir) cell bodies were detected in the posterior part of the hypothalamus. In the pituitary, a small number of PrRP-ir fibers were observed adjacent to the prolactin cells, whereas numerous PrRP-ir fibers were detected not only in the hypothalamus but also widely throughout the brain. PrRP-ir cell bodies and prolactin cells were already detected on the birth day in the hypothalamus and pituitary, respectively. The number of PrRP-ir fibers in the brain increased as the fish developed. These results suggest that PrRP is involved in neuromodulation in the brain and that PrRP plays some physiological roles in the early development of the guppy.

Immunohistochemical localization of orexin/hypocretin-like immunoreactive peptides and melanin-concentrating hormone in the brain and pituitary of medaka.

Orexin/hypocretin is a neuropeptide that is involved in the regulation of feeding behavior and the sleep-wakefulness cycle in mammals. Melanin-concentrating hormone (MCH) is believed to be another candidate involved in food intake in teleost fish as well. Thus, it is interesting to examine whether neural connections exist between the neurons producing these two hormones. We first examined the localization of orexin-like immunoreactivity (orexin-LI) in the brain of the medaka Oryzias latipes by using immunohistochemistry. We further examined the interaction between the orexin and MCH neurons in the medaka brain by performing double-staining immunohistochemistry. Orexin-LI cell bodies were located in the nucleus posterioris periventricularis (NPPv) of the hypothalamus, and orexin-LI fibers were detected not only in the hypothalamus but also extensively throughout the brain. Some orexin-LI fibers were in close contact with the MCH-immunoreactive (ir) cell bodies in the hypothalamus, as revealed by double-staining immunohistochemistry. Moreover, a few MCH-ir fibers were in close contact with the orexin-LI cell bodies. These results suggest that in the medaka brain, orexin performs various functions, including neuromodulation, and that neural connections exist between the orexin and MCH neurons.

Novel fish hypothalamic neuropeptides stimulate the release of gonadotrophins and growth hormone from the pituitary of sockeye salmon.

We recently identified a cDNA encoding three novel fish hypothalamic neuropeptides, having LPXRF-NH(2) from the goldfish brain. In this study, to clarify the physiological functions of these three LPXRFamide peptides (gfLPXRFa-1, -2, and -3), we analysed the localisation and hypophysiotrophic activity of these peptides using sockeye salmon, Oncorhynchus nerka, in which immunoassay systems for several anterior pituitary hormones have been developed. gfLPXRFa-immunoreactive cell bodies were detected in the nucleus posterioris periventricularis of the hypothalamus and immunoreactive fibres were distributed in various brain regions and the pituitary. We also detected gfLPXRFa-immunoreactivity in the pituitary by competitive enzyme-linked immunosorbent assay combined with reversed-phase HPLC. These three gfLPXRFamide peptides stimulated the release of FSH, LH and GH, but did not affect the release of prolactin (PRL) and somatolactin (SL) from cultured pituitary cells. These results suggest that novel fish hypothalamic LPXR-Famide peptides exist in the brain and pituitary of sockeye salmon and stimulate the release of gonadotrophins and GH from the pituitary.

A homolog of mammalian PRL-releasing peptide (fish arginyl-phenylalanyl-amide peptide) is a major hypothalamic peptide of PRL release in teleost fish.

Two PRL-releasing peptides (PrRP20 and PrRP31) were recently identified from mammalian hypothalamus by an orphan receptor strategy, and a C-terminal RF (arginyl-phenylalamyl-) amide peptide (RFa), structurally related to mammalian PrRP, was also identified from the brain of the Japanese crucian carp (C-RFa) by an intestine-contracting assay. However, to date there have been no reported studies that have examined the PRL-releasing effects of RFa in fish. In the present study we determined the cDNA, primary structure, and function of a homolog of the mammalian PrRP20 in the chum salmon, Oncorhynchus keta. An RFa cDNA encoding a preprohormone of 155 amino acids was cloned from the hypothalamus of chum salmon by 3'- and 5'-rapid amplification of cDNA ends. A native RFa was purified from an acid extract of salmon hypothalami by a Sep-Pak C(18) cartridge, affinity chromatography using anti-synthetic C-RFa, and reverse phase HPLC on an ODS-120T column. The salmon RFa proved to be identical with C-RFa on the basis of elution position on reverse phase HPLC. Immunocytochemical staining in rainbow trout, Oncorhynchus mykiss, revealed that C-RFa-immunoreactive cell bodies were located in the posterior part of hypothalamus and C-RFa-immunoreactive fibers were abundant from the hypothalamus to the ventral telencephalon. A small number of immunoreactive fibers were projected to the pituitary and terminated close to the PRL cells in the rostral pars distalis and to the somatolactin (SL) cells in the pars intermedia. The hypophysiotropic effects of the fish homolog were determined on the release of PRL, SL, and GH from the pituitary of the rainbow trout. Plasma PRL and SL levels were increased at 3 and 9 h, respectively, after ip injection of the synthetic C-RFa into the rainbow trout at doses of 50 and 500 ng/g body weight. In contrast, plasma GH levels were decreased after 1 h at 500 ng/g body weight. Perifusion of the trout pituitaries with synthetic C-RFa at concentrations of 10 pM to 100 nM demonstrated maximum PRL release at 100 pM and maximum SL release at 10 and 100 nM. However, GH release was not affected. These data are the first to demonstrate that a homolog of mammalian PrRP (fish RFa) is a major hypothalamic peptide of PRL release in teleost fish.

Possible involvement of melanin-concentrating hormone in food intake in a teleost fish, barfin flounder.

We investigated the involvement of MCH in food intake in barfin flounder. The structure of barfin flounder MCH was determined by cDNA cloning and mass spectrometry. In fasted fish, the MCH gene expression and the number of MCH neurons in the brain were greater than controls. In white-reared fish, the MCH gene expression and the number of MCH neurons in the brain were greater than black-reared fish. Furthermore, white-reared fish grew faster than black-reared fish. These results indicate that a white background stimulated production of MCH and MCH, in turn, enhanced body growth, probably by stimulating food intake.

Daily variations in melatonin binding sites in the masu salmon brain.

Daily variations in melatonin binding sites in the brain of underyearling masu salmon Oncorhynchus masou were examined by radioreceptor assay using 2-[125I]iodomelatonin as the radioligand. Fish were reared under a natural photoperiod in July and sampled eight times at 3 h intervals from 12:00 to 09:00 h. Plasma melatonin levels showed robust daily rhythms in both precocious males and immature females, with high and low levels during night and day, respectively. The affinity (Kd) and density (Bmax) of melatonin binding sites in the brain also showed similar variations. There were significant positive correlations between the plasma melatonin levels and the Kd or the Bmax in immature females and between the Kd and Bmax values in both precocious males and immature females. These results indicate that melatonin binding sites in the brain showed daily variations under a natural photoperiod in masu salmon.

Occurrence of saxitoxins as a major toxin in the ovary of a marine puffer Arothron firmamentum.

Eleven male and 14 female specimens of a marine puffer Arothron firmamentum were collected from Oita and Iwate Prefectures, Japan. The toxicity assay using mouse showed that only ovary and skin of the female specimens were toxic, the toxicity scores being 5-740 as paralytic shellfish poison and <5-30 MU/g as tetrodotoxin (TTX), respectively. The toxin extracts from the both tissues were then treated with cartridge columns, and subjected to high performance liquid chromatography and liquid chromatography-mass spectral analyses. In the analyses, saxitoxin (STX) and decarbamoylSTX (dcSTX) were identified as the major toxins in the ovary, while the skin contained only TTX.

Ontogenic development of three GnRH systems in the brain of a pleuronectiform fish, barfin flounder.

A pleuronectiform fish, the barfin flounder Verasper moseri, has three molecular forms of gonadotropin-releasing hormone (GnRH) in the brain, salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II) and seabream GnRH (sbGnRH). To elucidate the ontogenic origin of the neurons that produce these GnRH molecules, the development of three GnRH systems was examined by in situ hybridization and immunocytochemistry. Neuronal somata that express sGnRH mRNA were detected first in the vicinity of the olfactory epithelium 21 days after hatching (Day 21), and then in the transitional area between the olfactory nerve and olfactory bulb and the terminal nerve ganglion on Day 28. cGnRH-II mRNA-expressing neuronal somata were first identified in the midbrain tegmentum near the ventricle on Day 7. cGnRH-II-immunoreactive (ir) fibers were first found in the brain on Day 7. sbGnRH mRNA-expressing neuronal somata were first detected in the preoptic area on Day 42. sbGnRH-ir fibers were localized in the preoptic area-hypothalamus, and formed a distinctive bundle of axons projecting to the pituitary on Day 70. These results indicate that three forms of GnRH neurons have separate embryonic origins in the barfin flounder as in other perciform fish such as tilapia Oreochromis niloticus and red seabream Pagrus major: sGnRH, cGnRH-II and sbGnRH neurons derive from the olfactory placode, the midbrain tegmentum near the ventricle and the preoptic area, respectively.

Disturbance of plasma melatonin profile by high dose melatonin administration inhibits testicular maturation of precocious male masu salmon.

We have previously shown that the testicular development of underyearling male masu salmon Oncorhynchus masou reared under a long photoperiod was accelerated by oral melatonin treatment (0.5 mg melatonin/kg body weight/day), suggesting that melatonin mediates photoperiodic signaling. In this study, we further examined the effects of a disturbance in the plasma melatonin profile on gonadal development in underyearling male masu salmon by administering a higher dose of melatonin. Fish randomly selected in June were divided into two groups. They were reared under a light:dark (LD) cycle of 16:8 (lights on 04:00-20:00 hr) and fed with pellets sprayed with melatonin or vehicle twice a day at 08:30 and at 15:30 hr (7.5 mg melatonin/kg body weight/day) until October. Fish were sampled on Day 0, 25, 60, 90 and 120. The plasma melatonin levels were high in the dark phase and low in the light phase in the control group, while they were constantly high with no significant change in the melatonin-treated group. Melatonin treatment had inhibitory effects on the gonadosomatic index and plasma testosterone levels. Pituitary salmon gonadotropin-releasing hormone content and luteinizing hormone content were significantly lower in the melatonin-treated group on Day 60 and 90, respectively. These results indicate that the plasma melatonin profile is important for mediating photoperiodic signals that regulate brain-pituitary-gonadal axis in underyearling precocious male masu salmon.

Changes in brain GnRH mRNA and pituitary GnRH peptide during testicular maturation in barfin flounder.

The pleuronectid barfin flounder (Verasper moseri) expresses three forms of gonadotropin-releasing hormone (GnRH) in the brain. To clarify the physiological roles of the respective forms during testicular maturation, changes in brain GnRH mRNA levels and pituitary GnRH peptide levels were examined by real-time quantitative PCR and time-resolved fluoroimmunoassay, respectively. Fish hatched in April 2000. The gonadosomatic index remained low until October 2001 and then rapidly increased in January 2002. Fish continued to grow from hatching through testicular maturation. Fish spermiated in March 2002. The amount of seabream GnRH (sbGnRH) mRNA per brain significantly increased in January 2002 and remained at high levels in March 2002. The amounts of salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II) mRNA per brain did not show significant changes during the experimental periods. Pituitary sbGnRH peptide content significantly increased in March 2002. Pituitary sGnRH peptide and cGnRH-II peptide contents were extremely low compared to sbGnRH peptide levels and showed no significant changes during the experiment. These results indicate that sbGnRH is involved in the testicular maturation of barfin flounder.

Variety in histochemical characteristics of the olfactory receptor cells in a flatfish, barfin flounder (Verasper moseri).

Variety in histochemical characteristics of the olfactory receptor cells (ORC) was examined by immunohistochemistry for protein gene product 9.5 (PGP9.5) and calretinin, and by lectin histochemistry with Phaseolus vulgaris leucoagglutinin (PHA-L) in the olfactory epithelium (OE) of the barfin flounder (Verasper moseri). PGP 9.5 immunoreactivity was observed in the ORC situated in the upper three fourths of the OE. Calretinin immunoreactivity was observed in the ORC which seemed to be immunonegative for PGP 9.5. These cells were located in the upper two thirds of the OE. PHA-L staining was observed in small subsets of the ORC. PGP 9.5 and calretinin immunoreactivities and PHA-L staining were also observed in the crypt cells unique to the fish OE. These findings suggest the different properties of olfactory perception among fish ORC.

Morphogenesis of the olfactory pit in a flatfish, barfin flounder (Verasper moseri).

Morphogenesis of the olfactory pit (OP), olfactory lamella (OL) and olfactory epithelium (OE) was examined by scanning electron and light microscopy in the barfin flounder (Verasper moseri). At day 0 after hatch, the OP was already formed. At day 14, the cellular differentiation of the OE was prominent. At day 42, the OP became a cavity by the formation of its roof. At day 56, the first OL extended remarkably and was lined with the OE on both sides. The OL increased in number with development. These findings suggest that the OE is functionally active at day 14. The formation of the OL in the OP may be initiated by the stimulus when the barfin flounder touched at the bottom of the sea.

Differential expression of histochemical characteristics in the developing olfactory receptor cells in a flatfish, barfin flounder (Verasper moseri).

Differentiation of the histochemical characteristics of the olfactory receptor cells (ORC) was examined by immunohistochemistry for protein gene product 9.5 (PGP 9.5) and calretinin (CR) and lectin histochemistry for Phaseolus vulgaris agglutinin-L (PHA-L) in the developing olfactory epithelium (OE) of the barfin flounder. PGP 9.5 immunoreactivity was diffuse and CR immunoreactivity was restricted at day 7, but these immunoreactivities became intense in the OE toward day 91. Crypt cells were first identified at day 56. PHA-L staining was faint at day 28, but became intense toward day 91. These findings suggest that PGP 9.5-immunopositive cells, CR-immunopositive cells, crypt cells and PHA-L-reactive cells differentiate independently in the developing OE and constitute subsets of the ORC in the OE.

[The toxification of juvenile cultured kusafugu Takifugu niphobles by oral administration of crystalline tetrodotoxin].

Non-toxic cultured juvenile kusafugu were fed with diet containing crystalline tetrodotoxin (TTX) for 30 days and then fed with non-toxic diet for 170 days. During this period, 5 fish were sampled and the toxicity of each tissue was determined periodically. The amount of total accumulated toxin in the fish was 90 microg, representing 50% of the administered TTX (180 microg/fish) at the 60th day. It decreased to 54 microg (30%) at the 80th day and then remained unchanged up to the 200th day. The amount of toxin in the liver amounted to 40 microg (45% of total accumulated toxin) at the 30th day and gradually decreased to 5 microg (10%) at the 200th day. The toxin amount in the skin reached the highest level with 30 microg (30%) at the 50th day and then remained unchanged during the experimental period. The testes had almost no toxicity. Although the ovaries were immature, the toxin amounts increased as the weight of the tissues increased. With administration of crystalline TTX, all kusafugu used in the experiment became toxic and retained the toxin at the level of 30% of the administered toxin for about 5 months thereafter, while being fed with non-toxic diet.

Immunohistochemical localization of a GnRH-like peptide in the brain of the cephalopod spear-squid, Loligo bleekeri.

We examined whether a gonadotropin-releasing hormone (GnRH)-like peptide exists in the brain of the cephalopod spear-squid, Loligo bleekeri, by performing a time-resolved fluoroimmunoassay and immunohistochemistry. The displacement curve obtained for serially diluted extracts of the spear-squid brain paralleled the chicken GnRH-II (cGnRH-II) standard curve, indicating the existence of a cGnRH-II-like peptide in the brain. For immunohistochemistry, a mouse monoclonal antibody raised against the common amino acid sequence of GnRH (LRH13) and a rabbit polyclonal antibody raised against cGnRH-II were used. GnRH-like-immunoreactive (ir) cell bodies (that reacted with LRH13) were mainly detected in the central part of the ventral magnocellular lobe (vmL), and a few cell bodies were also detected in the olfactory lobe and palliovisceral lobe (pvL). Bundles of GnRH-like-ir axons were observed running from the vmL to the internal brain regions. GnRH-like-ir fibers were widely distributed in almost all the brain regions. cGnRH-II-ir cell bodies were localized in the optic gland, outer region of the vmL, and pvL. Further, cGnRH-II-ir fibers were distributed in the wide areas of the brain. These results suggest that at least two forms of GnRH-like peptidergic neuronal systems exist in the spear-squid brain.