Lunar Age-Dependent Oscillations in Expression of the Genes for Kisspeptin, GnIH, and Their Receptors in the Grass Puffer during the Spawning Season.

Grass puffer is a semilunar-synchronized spawner: spawning occurs on beaches only for several days of spring tide around new moon (lunar age 0) and full moon (lunar age 15) every 2 weeks from spring to early summer. To investigate the role of kisspeptin and gonadotropin-inhibitory hormone (GnIH) in the semilunar-synchronized spawning, lunar age-dependent expression of the genes encoding kisspeptin (kiss2), kisspeptin receptor (kissr2), GnIH (gnih), GnIH receptor (gnihr), gonadotropin-releasing hormone 1 (GnRH1) (gnrh1), and three gonadotropin (GTH) subunits (gpa, fshb, lhb) was examined in the male grass puffer, which was kept in an aquarium under natural light condition in a lunar month during the spawning period. In the brain, both kiss2 and kissr2 showed lunar variations with a peak at lunar age 10, while both gnih and gnihr showed semilunar variations with two peaks at lunar age 0 and 20. On the other hand, gnrh1 showed semilunar variation with two peaks at lunar age 0 and 15. In the pituitary, kiss2, kissr2, gnih, and gnihr showed similar variations to those shown in the brain. The fshb and lhb mRNA levels showed semilunar variations with two peaks at lunar age 0 and 15. The present study shows lunar and semilunar oscillations of kiss2/kissr2 and gnih/gnihr expressions, respectively, with their peaks around spring tide in the brain and pituitary along with the semilunar expressions of gnrh1 and the pituitary GTH subunit genes. These results suggest that the lunar age-dependent expressions of the kisspeptin, GnIH, and their receptor genes may be primarily important in the control of the precisely timed semilunar spawning of the grass puffer.

Two cases of systemic lupus erythematosus after administration of severe acute respiratory syndrome coronavirus 2 vaccine.

Although there is a great demand for increased coronavirus disease 2019vaccination worldwide, rare side effects of the vaccines in susceptible individuals are attracting attention. We recently treated two patients who developed systemic lupus erythematosus after administration of a severe acute respiratory syndrome coronavirus 2 vaccine from Pfizer-BioNTech or Moderna. While causal relationships between vaccination and adverse events are difficult to discern due to both confounding and masking factors, our findings suggest that attention to possible adjuvant-related autoimmune diseases in certain individuals receiving severe acute respiratory syndrome coronavirus 2 vaccines is appropriate.

Prostaglandin E2 synchronizes lunar-regulated beach spawning in grass puffers.

Many organisms living along the coastlines synchronize their reproduction with the lunar cycle. At the time of spring tide, thousands of grass puffers (Takifugu alboplumbeus) aggregate and vigorously tremble their bodies at the water's edge to spawn. To understand the mechanisms underlying this spectacular semilunar beach spawning, we collected the hypothalamus and pituitary from male grass puffers every week for 2 months. RNA sequencing (RNA-seq) analysis identified 125 semilunar genes, including genes crucial for reproduction (e.g., gonadotropin-releasing hormone 1 [gnrh1], luteinizing hormone ß subunit [lhb]) and receptors for pheromone prostaglandin E (PGE). PGE2 is secreted into the seawater during the spawning, and its administration activates olfactory sensory neurons and triggers trembling behavior of surrounding individuals. These results suggest that PGE2 synchronizes lunar-regulated beach-spawning behavior in grass puffers. To further explore the mechanism that regulates the lunar-synchronized transcription of semilunar genes, we searched for semilunar transcription factors. Spatial transcriptomics and multiplex fluorescent in situ hybridization showed co-localization of the semilunar transcription factor CCAAT/enhancer-binding protein δ (cebpd) and gnrh1, and cebpd induced the promoter activity of gnrh1. Taken together, our study demonstrates semilunar genes that mediate lunar-synchronized beach-spawning behavior. VIDEO ABSTRACT.

Kisspeptin Exhibits Stimulatory Effects on Expression of the Genes for Kisspeptin Receptor, GnRH1 and GTH Subunits in a Gonadal Stage-Dependent Manner in the Grass Puffer, a Semilunar-Synchronized Spawner.

Kisspeptin has an important role in the regulation of reproduction by directly stimulating the secretion of gonadotropin-releasing hormone (GnRH) in mammals. In non-mammalian vertebrates, there are multiple kisspeptins (Kiss1 and Kiss2) and kisspeptin receptor types, and the two kisspeptins in teleosts have different effects depending on fish species and reproductive stages, serving reproductive and non-reproductive functions. In the grass puffer, Takifugu alboplumbeus, which has only a single pair of kiss2 and kissr2, both genes display seasonal, diurnal, and circadian oscillations in expression in association with the periodic changes in reproductive functions. To elucidate the role of kisspeptin in this species, homologous kisspeptin peptide (gpKiss2) was administered at different reproductive stages (immature, mature and regressed) and the expression levels of the genes that constitute hypothalamo-pituitary-gonadal axis were examined in male grass puffer. gpKiss2 significantly elevated the expression levels of kissr2 and gnrh1 in the brain and kissr2, fshb and lhb in the pituitary of the immature and mature fish. No noticeable effect was observed for kiss2, gnih, gnihr, gnrh2 and gnrh3 in the brain and gpa in the pituitary. In the regressed fish, gpKiss2 was ineffective in stimulating the expression of the gnrh1 and GTH subunit genes, while it stimulated and downregulated the kissr2 expression in the brain and pituitary, respectively. The present results indicate that Kiss2 has a stimulatory role in the expression of GnRH1/GTH subunit genes by upregulating the kissr2 expression in the brain and pituitary at both immature and mature stages, but this role is mostly ineffective at regressed stage in the grass puffer.

Anomalous Temperature Interdicts the Reproductive Activity in Fish: Neuroendocrine Mechanisms of Reproductive Function in Response to Water Temperature.

Fish are poikilotherm and small changes in water temperature can greatly affect physiological processes including reproduction, which is regulated by complex neuroendocrine mechanisms that respond to climatic events. This review provides evidence that anomalous high and low temperature may directly affect reproduction in fish by suppressing the expression of genes in the reproductive neuroendocrine system. The grass puffer, Takifugu alboplumbeus, is an excellent animal model for studying the thermal regulation of reproduction, for they exhibit periodic spawning activities, which are synchronized with seasonal, lunar and daily cycles. In the grass puffer, the expression of the genes encoding gonadotropin-releasing hormone (GnRH) 1, kisspeptin, gonadotropin-inhibitory hormone (GnIH) and their receptors were markedly suppressed in the diencephalon of fish exposed to high temperature (28°C) when compared to normal temperature (21°C), followed by the decrease in the pituitary mRNA levels for follicle-stimulating hormone (FSH), luteinizing hormone (LH) and growth hormone (GH). On the other hand, the exposure to low temperature (14°C) also inhibited the expression of gnrh1, kiss2, gnih and their receptor genes in the brain and fshb, lhb, gh and prl in the pituitary. Taken together, it is plausible that anomalous high and low temperature may be a proximate driver of termination of reproduction by suppressing the activity of the reproductive GnRH/kisspeptin/GnIH system, possibly through direct action of temperature signals at transcription level.

Higher acclimation temperature affects growth of rohu (Labeorohita) through suppression of GH and IGFs genes expression actuating stress response.

Water temperature alone can affect the growth, metabolic rates and physiological responses of aquatic organisms. Our earlier study reported that higher temperature affects cellular and hemato-biochemical responses in rohu, Labeo rohita. In this backdrop, the present study assessed the effect of higher acclimation temperature on the regulatory mechanisms of growth and stress responses of juvenile L. rohita acclimatized in three temperature conditions (30 °C, 33 °C, and 36 °C) for a period of 30 days. The relative expression of genes for growth hormone (GH), insulin-like growth factors (IGF-1 and IGF-2) and heat shock proteins (hsp70 and hsp90) were measured by real-time quantitative PCR. The results revealed that the highest acclimation temperature (36 °C) significantly decreased the weight gain (WG) and specific growth rate (SGR), and increased the feed conversion ratio (FCR) compared to 30 °C (control), while increased WG, SGR and lowered FCR were observed in fish reared at the intermediate temperature (33 °C) compared to 30 °C. Similarly, the GH gene expression in the pituitary was significantly decreased and increased at 36 °C and 33 °C, respectively as compared to 30 °C. A significantly lower expression of IGF-1 and IGF-2, and higher expression of hsp70 and hsp90 were observed in the liver of fish at 36 °C. The results of the present study indicate that although slightly elevated temperature promotes the growth of juvenile L. rohita, the higher acclimation temperature may induce stress response and impair growth performance by suppressing GH/IGF system.

Light-induced and circadian expressions of melanopsin genes opn4xa and opn4xb in the eyes of juvenile grass puffer Takifugu alboplumbeus.

Animals regulate a variety of aspects of physiology according to environmental light conditions via nonvisual opsins such as melanopsin. In order to study photic regulation of fish physiology, expression changes of the genes for melanopsin (opn4xa and opn4xb) and effects of light on them were examined in juvenile grass puffer Takifugu alboplumbeus using quantitative real-time PCR. In the brain of juvenile fish, no significant diurnal nor circadian changes were observed in opn4x mRNA levels. On the other hand, in the eyes, the mRNA level of opn4xa showed a significant diurnal rhythm with a peak at Zeitgeber time (ZT) 4, while no apparent circadian changes were observed. The mRNA level of opn4xb in the eyes showed a diurnal change similar to that of opn4xa, while it showed a significant circadian change. Furthermore, continuous exposure to light during a subjective night significantly increased the mRNA levels of opn4xa in the eyes at ZT24, suggesting that light induces gene expression of opn4xa in the eyes and that the induction occurs only during the night-day transition period. These results suggest that Opn4xa and Opn4xb play differential roles in the eyes of juvenile grass puffer to mediate the physiological effects of environmental light information.

Expression dynamics of the genes for the hypothalamo-pituitary-gonadal axis in tiger puffer (Takifugu rubripes) at different reproductive stages.

Tiger puffer, Takifugu rubripes, a commercially important long-distance migratory fish, return to specific spawning grounds for reproduction. To clarify reproductive neuroendocrine system of the tiger puffer, the changes in the expression levels of the genes encoding three gonadotropin-releasing hormones (GnRHs), gonadotropin-inhibitory hormone (GnIH), GnIH receptor (GnIH-R), kisspeptin and kisspeptin receptor in the brain and gonadotropin (GTH) subunits, growth hormone (GH) and prolactin (PRL) in the pituitary were examined in the tiger puffer captured in the wild at different reproductive stages, namely immature and mature fish of both sexes, and post-ovulatory females that were obtained by hormonal treatment. The amounts of three gnrh mRNAs, gnih, gnih-r, fshb and lhb were substantially increased in the mature fish compared to the immature fish, especially in the females, and these augmented expressions were drastically decreased in the post-ovulatory females. gh expression showed a slight increase in the mature males. In contrast, kiss2, kiss2r and prl did not show significant changes in the males but significantly decreased in the post-ovulatory females. The present results demonstrate the expression dynamics of the hypothalamo-pituitary-gonadal axis genes associated with the reproductive conditions and the possible involvement of the GnRH/GnIH/GTH system in the regulation of the sexual maturation and spawning in the wild tiger puffer.

Melatonin is a potential drug for the prevention of bone loss during space flight.

Astronauts experience osteoporosis-like loss of bone mass because of microgravity conditions during space flight. To prevent bone loss, they need a riskless and antiresorptive drug. Melatonin is reported to suppress osteoclast function. However, no studies have examined the effects of melatonin on bone metabolism under microgravity conditions. We used goldfish scales as a bone model of coexisting osteoclasts and osteoblasts and demonstrated that mRNA expression level of acetylserotonin O-methyltransferase, an enzyme essential for melatonin synthesis, decreased significantly under microgravity. During space flight, microgravity stimulated osteoclastic activity and significantly increased gene expression for osteoclast differentiation and activation. Melatonin treatment significantly stimulated Calcitonin (an osteoclast-inhibiting hormone) mRNA expression and decreased the mRNA expression of receptor activator of nuclear factor κB ligand (a promoter of osteoclastogenesis), which coincided with suppressed gene expression levels for osteoclast functions. This is the first study to report the inhibitory effect of melatonin on osteoclastic activation by microgravity. We also observed a novel action pathway of melatonin on osteoclasts via an increase in CALCITONIN secretion. Melatonin could be the source of a potential novel drug to prevent bone loss during space flight.

Effects of high and low temperature on expression of GnIH, GnIH receptor, GH and PRL genes in the male grass puffer during breeding season.

Gonadotropin-inhibitory hormone (GnIH) is a multifunctional hypophysiotropic neurohormone and has a stimulatory role in the control of reproduction in the grass puffer. To clarify the neuroendocrine mechanisms underlying the effect of changes in water temperature on reproduction in fish, we previously revealed that, in parallel to gonadal regression, both low and high temperature significantly decreased the expressions of the genes encoding kisspeptin (kiss2), kisspeptin receptor (kiss2r), gonadotropin-releasing hormone 1 (gnrh1) in the brain and gonadotropin (GTH) subunits (fshb and lhb) in the pituitary of sexually mature male grass puffer. In this study, we examined the changes in expression of gnih and GnIH receptor gene (gnihr) in the brain and pituitary along with the genes for growth hormone (gh) and prolactin (prl) in the pituitary of male grass puffer exposed to low temperature (14 °C), normal temperature (21 °C, as initial control) and high temperature (28 °C) conditions for 7 days. The levels of gnih and gnihr mRNAs were significantly decreased in both low and high temperature conditions compared to normal temperature in the brain and pituitary. Similarly, the gh mRNA levels were significantly decreased in both low and high temperature conditions. The prl mRNAs showed no significant changes at high temperature, whereas drastically decreased at low temperature possibly by dysfunctional cold stress. Taken together, the present results suggest that, in addition to the inhibitory effect of temperature changes on the Kiss2/GnRH1/GTH system, the suppression of GnIH/GH system may also be involved in the termination of reproduction by high temperature at the end of breeding season.

Periodic regulation of expression of genes for kisspeptin, gonadotropin-inhibitory hormone and their receptors in the grass puffer: Implications in seasonal, daily and lunar rhythms of reproduction.

The seasonal, daily and lunar control of reproduction involves photoperiodic, circadian and lunar changes in the activity of kisspeptin, gonadotropin-inhibitory hormone (GnIH) and gonadotropin-releasing hormone (GnRH) neurons. These changes are brought through complex networks of light-, time- and non-photic signal-dependent control mechanisms, which are mostly unknown at present. The grass puffer, Takifugu alboplumbeus, a semilunar spawner, provides a unique and excellent animal model to assess this question because its spawning is synchronized with seasonal, daily and lunar cycles. In the diencephalon, the genes for kisspeptin, GnIH and their receptors showed similar expression patterns with clear seasonal and daily oscillations, suggesting that they are regulated by common mechanisms involving melatonin, circadian clock and water temperature. For implications in semilunar-synchronized spawning rhythm, melatonin receptor genes showed ultradian oscillations in expression with the period of 14.0-15.4 h in the pineal gland. This unique ultradian rhythm might be driven by circatidal clock. The possible circatidal clock and circadian clock in the pineal gland may cooperate to drive circasemilunar rhythm to regulate the expression of the kisspeptin, GnIH and their receptor genes. On the other hand, high temperature (over 28 °C) conditions, under which the expression of the kisspeptin and its receptor genes is markedly suppressed, may provide an environmental signal that terminates reproduction at the end of breeding period. Taken together, the periodic regulation of the kisspeptin, GnIH and their receptor genes by melatonin, circadian clock and water temperature may be important in the precisely-timed spawning of the grass puffer.

Production of recombinant salmon insulin-like growth factor binding protein-1 subtypes.

Insulin-like growth factor (IGF)-I is a growth promoting hormone that exerts its actions through endocrine, paracrine and autocrine modes. Local IGF-I is essential for normal growth, whereas circulating IGF-I plays a crucial role in regulating the production and secretion of growth hormone (GH) by the pituitary gland. These actions of IGF-I are modulated by six insulin-like growth factor binding proteins (IGFBPs). In teleosts, two subtypes of each IGFBP are present due to an extra round of whole-genome duplication. IGFBP-1 is generally inhibitory to IGF-I action under catabolic conditions such as fasting and stress. In salmon, IGFBP-1a and -1b are two of three major circulating IGFBPs and assumed to affect growth through modulating IGF-I action. However, exact functions of salmon IGFBP-1 subtypes on growth regulation are not known due to the lack of purified or recombinant protein. We expressed recombinant salmon (rs) IGFBP-1a and -1b with a fusion protein (thioredoxin, Trx) and a His-tag using the pET-32a(+) vector expression system in Escherichia coli. Trx.His.rsIGFBP-1s were isolated by Ni-affinity chromatography, enzymatically cleaved by enterokinase to remove the fusion partners and further purified by reversed-phase HPLC. We next examined effects of rsIGFBP-1a and -1b in combination with human IGF-I on GH release from cultured masu salmon (Oncorhynchus masou) pituitary cells. Unexpectedly, IGF-I increased GH release and an addition of rsIGFBP-1a, but not rsIGFBP-1b, restored GH levels. The results suggest that IGFBP-1a can inhibit IGF-I action on the pituitary in masu salmon. Availability of recombinant salmon IGFBP-1s should facilitate further functional analyses and assay development.

Temperature affects sexual maturation through the control of kisspeptin, kisspeptin receptor, GnRH and GTH subunit gene expression in the grass puffer during the spawning season.

Water temperature is an environmental factor of primary importance that influences reproductive function in fish. To understand the molecular and physiological mechanisms underlying the regulation of reproduction by temperature, we examined changes in expression of genes encoding kisspeptin (kiss2), kisspeptin receptor (kiss2r) and three gonadotropin-releasing hormones (gnrh1, gnrh2 and gnrh3) in the brain and genes encoding gonadotropin (GTH) subunits (gpa, fshb and lhb) in the pituitary of grass puffer exposed to a low temperature (14°C), normal temperature (21°C) and high temperature (28°C) for 7days. In addition, the plasma levels of cortisol were examined after exposed to three temperature conditions. The gonadosomatic index was significantly decreased in both low and high temperature conditions. The levels of kiss2 and kiss2r mRNAs were significantly decreased at both low and high temperature conditions compared to normal temperature (control) condition. gnrh1 but not gnrh2 were significantly decreased in both temperature conditions, while gnrh3 showed a decreasing tendency in low temperature. Consequently, the levels of fshb and lhb mRNAs were significantly decreased in both low and high temperature conditions. Interestingly, the plasma levels of cortisol were significantly increased in low temperature but remain unchanged in high temperature, suggesting that the fish were under stress in the low temperature conditions but not in the high temperature conditions. Taken together, the present results indicate that anomalous temperature have an inhibitory effect on reproductive function through suppressing kiss2/kiss2r/gnrh1/fshb and lhb expression and these changes may occur in a normal physiological response as well as in a malfunctional stress response.

LPXRFamide peptide stimulates growth hormone and prolactin gene expression during the spawning period in the grass puffer, a semi-lunar synchronized spawner.

Gonadotropin-inhibitory hormone (GnIH) plays as a multifunctional neurohormone that controls reproduction in birds and mammals. LPXRFamide (LPXRFa) peptide, the fish ortholog of GnIH, has been shown to regulate the secretion of not only gonadotropin (GTH) but also growth hormone (GH) and prolactin (PRL), which are potentially important for gonadal function. To investigate the role of LPXRFa peptide on reproduction of the grass puffer, which spawns in semilunar cycles, we examined changes in the levels of gh and prl expression over the several months during the reproductive cycle, and the effects of goldfish LPXRFa peptide-1 (gfLPXRFa-1) on their expression were examined using primary pituitary cultures. The expression levels of both gh and prl showed significant changes during the reproductive cycle in both sexes with one peak in the spawning and pre-spawning periods for gh and prl, respectively. Particularly, gh showed substantial increase in expression in the spawning and post-spawning periods, indicative of its essentiality in the advanced stage of reproduction. gfLPXRFa-1 stimulated the expression of both gh and prl but there was a marked difference in response between them: gfLPXRFa-1 stimulated gh expression at a relatively low dose but little effect was observed on prl. Combined with the previous results of daily and circadian oscillations of lpxrfa expression, the present results suggest that LPXRFa peptide is important in the control of the cyclic reproduction by serving as a multifunctional hypophysiotropic factor that regulates the expression of gh and prl as well as GTH subunit genes.

Ultradian oscillation in expression of four melatonin receptor subtype genes in the pineal gland of the grass puffer, a semilunar-synchronized spawner, under constant darkness.

Melatonin receptor gene expression as well as melatonin synthesis and secretion activities were examined in the pineal gland of the grass puffer, which exhibits unique lunar/tidal cycle-synchronized mass spawing: spawning occurs before high tide on the day of spring tide during spawing season. Melatonin synthesizing activity was assessed by the abundance of arylalkylamine N-acetyltransferase 2 (AANAT2) mRNA. The amount of aanat2 mRNA was low during light phase and initiated to increase after the light was turned off. The secretion of melatonin from primary pineal organ culture was stimulated after the light was turned off and ceased immediately after the light was turned on. The expression levels of four melatonin receptor subtype genes (mel 1a 1.4, mel 1a 1.7, mel1b, and mel1c) showed synchronous variations, and the levels tended to be high during the dark phase under light/dark conditions. These results suggest that the action of melatonin on the pineal gland is highly dependent on light and photoperiod, possibly with stronger action during night time. Under constant darkness, the expression of four melatonin receptor subtype genes showed unique ultradian oscillations with the period of 14.0-15.4 h, suggesting the presence of a circatidal oscillator in the pineal gland. The present results indicate that melatonin may serve local chronobiological functions in the pineal gland. These cyclic expressions of melatonin receptor genes in the pineal gland may be important in the control of the lunar/tidal cycle-synchronized mass spawning in the grass puffer.

Differential expression patterns of PQRFamide peptide and its two receptor genes in the brain and pituitary of grass puffer during the reproductive cycle.

Pain-modulatory neuropeptides, PQRFamide (PQRFa) peptides, have recently been implicated in the regulation of reproduction in fish. As a first step toward investigating the role of PQRFa peptides on reproductive function in the grass puffer Takifugu niphobles, which is a semilunar spawner, we cloned genes encoding PQRFa peptide precursor (pqrfa) and its two types of receptors (pqrfa-r1 and pqrfa-r2), and examined changes in their expression levels in the brain and pituitary over several months during the reproductive cycle. The grass puffer PQRFa peptide precursor of 126 amino acid residues contains two putative PQRFa peptides, PQRFa-1 and PQRFa-2, which correspond to NPFF and NPAF in other vertebrates, respectively. The grass puffer PQRFa-R1 and PQRFa-R2 consist of 426 and 453 amino acid residues, respectively, and contain distinct characteristics of G-protein coupled receptors. These three genes were exclusively expressed in the brain and pituitary. The expression levels of pqrfa and pqrfa-r1 were significantly increased during the late stage of sexual maturation, but low in the spawning fish just after releasing sperms and eggs. Therefore, the grass puffer PQRFa peptide may have a role in the late stage of sexual maturation before spawning via PQRFa-R1. In contrast, the pqrfa-r2 expression showed maximum levels in the spawning fish and in the post-spawning period. The present results provide fundamental data suggesting that the grass puffer PQRFa peptide may have multiple roles in the control of reproduction that are dependent on the reproductive stages.

Accumulation of organotins in wharf roach (Ligia exotica Roux) and its ability to serve as a biomonitoring species for coastal pollution.

In this study, we measured the accumulation of tributyltin (TBT) in wharf roach (Ligia exotica Roux) and examined the species' ability to be used for TBT biomonitoring in coastal environments. In an exposure test, wharf roach were exposed to TBT via diet for 2d. TBT was accumulated in wharf roach, and its metabolite dibutyltin was detected. The concentrations of these compounds gradually decreased during the depuration period, but they were still detected 12d after exposure ceased (TBT 290±140ng/g; dibutyltin 1280±430ng/g). The biological half-life of TBT in wharf roach was estimated to be about 4d. In a field study conducted in 2011-2012, wharf roach were collected from 15 coastal sites in Japan and 3 sites in Manado, Indonesia. TBT was detected in both Japanese and Indonesian samples. The highest concentration of TBT was found in wharf roach collected at Bitung ferry port, Manado (57.9±16.5ng/g), which is close to a shipyard, and the highest concentration at a Japanese site was 12.3±6.2ng/g. Thus, we were able to detect organotins in the coastal environments by testing wharf roach, suggesting that L. exotica might serve as a good bioindicator for monitoring organotin pollution.

Molecular neuroendocrine basis of lunar-related spawning in grass puffer.

Grass puffer, Takifugu niphobles, exhibits unique spawning behavior: it spawns on beach in semilunar cycles during spring tide in early summer. The fish aggregate at certain seashore locations several hours before high tide every two weeks. To explore the molecular and neuroendocrine mechanisms underlying the regulation of the lunar-related spawning rhythm, seasonal and cyclic variations in gene expression for hypothalamic neuropeptides related to reproduction were examined by quantitative real-time PCR. The expression levels of genes for gonadotropin-releasing hormone, kisspeptin, LPXRFamide peptide and PQRFamide peptide in the hypothalamus varied differently depending on reproductive stage and gender, suggesting their specific roles in reproduction. In the spawning period, the expression levels of LPXRFamide peptide and its receptor genes showed diurnal and circadian variations in association with the expression of four subtypes of melatonin receptor genes. Together with the nocturnal secretion of melatonin from the pineal gland, melatonin may play an important role in transmitting the photoperiodic information of moonlight to the reproductive neuroendocrine center in the hypothalamus of grass puffer.

Identification and gene expression analyses of ghrelin in the stomach of Pacific bluefin tuna (Thunnus orientalis).

Full length cDNA and gene encoding ghrelin precursor and mature ghrelin peptide were identified from the stomach of Pacific bluefin tuna, Thunnus orientalis, which has unique metabolic physiology and high commercial value at fishery markets. Quantitative expression analysis was conducted for the gastric ghrelin and pepsinogen 2 genes during the early stage of somatic growth from the underyearling to yearling fish. The full length cDNA of bluefin tuna ghrelin precursor has a length of 470bp and the deduced precursor is composed of 107 amino acids. The ghrelin gene is 1.9kbp in length and has a 4 exon-3 intron structure. The major form of mature ghrelin in the stomach was an octanoylated 20-amino acid peptide with C-terminal amidation, while overall 12 different forms of ghrelin peptides, including short form of 18-amino acid peptide and seven kinds of acyl modifications were identified. The expression profiles of the gastric ghrelin and pepsinogen 2 genes showed no significant changes related to the early growth stages. The present results suggest that digestive physiology has already been functional in this growth stage of the juvenile bluefin tuna and ghrelin may have a role in the sustained digestive and metabolic activities.