Stable Production of a Tethered Recombinant Eel Luteinizing Hormone Analog with High Potency in CHO DG44 Cells.

We produced a recombinant eel luteinizing hormone (rec-eel LH) analog with high potency in Chinese hamster ovary DG44 (CHO DG44) cells. The tethered eel LH mutant (LH-M), which had a linker comprising the equine chorionic gonadotropin (eLH/CG) ß-subunit carboxyl-terminal peptide (CTP) region (amino acids 115 to 149), was inserted between the ß-subunit and α-subunit of wild-type tethered eel LH (LH-wt). Monoclonal cells transfected with the tethered eel LH-wt and eel LH-M plasmids were isolated from five to nine clones of CHO DG44 cells, respectively. The secreted quantities abruptly increased on day 3, with peak levels of 5000-7500 ng/mL on day 9. The molecular weight of tethered rec-eel LH-wt was 32-36 kDa, while that of tethered rec-eel LH-M increased to approximately 38-44 kDa, indicating the detection of two bands. Treatment with the peptide N-glycanase F decreased the molecular weight by approximately 8 kDa. The oligosaccharides at the eCG ß-subunit O-linked glycosylation sites were appropriately modified post-translation. The EC50 value and maximal responsiveness of eel LH-M increased by approximately 2.90- and 1.29-fold, respectively, indicating that the mutant exhibited more potent biological activity than eel LH-wt. Phosphorylated extracellular regulated kinase (pERK1/2) activation resulted in a sharp peak 5 min after agonist treatment, with a rapid decrease thereafter. These results indicate that the new tethered rec-eel LH analog had more potent activity in cAMP response than the tethered eel LH-wt in vitro. Taken together, this new eel LH analog can be produced in large quantities using a stable CHO DG44 cell system.

Pinostrobin Suppresses the α-Melanocyte-Stimulating Hormone-Induced Melanogenic Signaling Pathway.

Pinostrobin is a dietary flavonoid found in several plants that possesses pharmacological properties, such as anti-cancer, anti-virus, antioxidant, anti-ulcer, and anti-aromatase effects. However, it is unclear if pinostrobin exerts anti-melanogenic properties and, if so, what the underlying molecular mechanisms comprise. Therefore, we, in this study, investigated whether pinostrobin inhibits melanin biosynthesis in vitro and in vivo, as well as the potential associated mechanism. Pinostrobin reduced mushroom tyrosinase activity in vitro in a concentration-dependent manner, with an IC50 of 700 µM. Molecular docking simulations further revealed that pinostrobin forms a hydrogen bond, as well as other non-covalent interactions, between the C-type lectin-like fold and polyphenol oxidase chain, rather than the previously known copper-containing catalytic center. Additionally, pinostrobin significantly decreased α-melanocyte-stimulating hormone (α-MSH)-induced extracellular and intracellular melanin production, as well as tyrosinase activity, in B16F10 melanoma cells. More specifically, pinostrobin inhibited the α-MSH-induced melanin biosynthesis signaling pathway by suppressing the cAMP-CREB-MITF axis. In fact, pinostrobin also attenuated pigmentation in α-MSH-stimulated zebrafish larvae without causing cardiotoxicity. The findings suggest that pinostrobin effectively inhibits melanogenesis in vitro and in vivo via regulation of the cAMP-CREB-MITF axis.

Effect of short- and long-term melatonin treatments on the reproductive activity of the tropical damselfish Chrysiptera cyanea.

Photoperiod plays a role in controlling the initiation and termination of reproduction in fish. Melatonin is an internal transducer of environmental photoperiod and is involved in regulating reproduction. The present study aimed to examine how melatonin impacts the transcript levels of kisspeptin (kiss1 and kiss2), gonadotropin-releasing hormones (gnrh1), and the ß-subunit of gonadotropins (fshß and lhß) in the brain of the sapphire devil, a tropical damselfish with long photoperiod preference. Feeding mature females with melatonin-containing pellets inhibited increases in the transcript levels of kiss1, gnrh1, and lhß within 3 h. Continuous melatonin treatment for 1 week resulted in oocyte regression and downregulation of kiss2, gnrh1, fshß, and lhß. When the transcript levels of kiss1 and gnrh1 were measured at 4-h intervals in the brain of sapphire devil, a day-high/night-low fluctuation was observed. The hypothalamic-pituitary-gonadal (HPG) axis may be influenced by melatonin, exerting a negative effect at night because the transcript levels of aralkylamine N-acetyltransferase (aanat2) increased during the scotophase. The expression of aanat2 was higher under short-day than long-day conditions, suggesting that there is a seasonal change in melatonin levels at night. It was concluded that change in photoperiod becomes a key factor for controlling the hormone synthesis in the HPG axis through melatonin.

Expression profiles of types 2 and 3 iodothyronine deiodinase genes in relation to vitellogenesis in a tropical damselfish, Chrysiptera cyanea.

Thyroid hormone (TH) is involved in regulating the reproduction of vertebrates. Its physiological action in the target tissues is due to the conversion of TH by iodothyronine deiodinases. In this study, we aimed to clone and characterize type 2 (sdDio2) and type 3 (sdDio3) of the sapphire devil Chrysiptera cyanea, a tropical damselfish that undergoes active reproduction under long-day conditions, and to study the involvement of THs in the ovarian development of this species. When the cDNAs of sdDio2 and sdDio3 were partially cloned, they had deduced amino acid sequences of lengths 271 and 267, respectively, both of which were characterized by one selenocysteine residue. Real-time quantitative PCR (qPCR) revealed that both genes are highly expressed in the whole brain, and sdDio2 and sdDio3 are highly transcribed in the liver and ovary, respectively. In situ hybridization analyses showed positive signals of sdDio2 and sdDio3 transcripts in the hypothalamic area of the brain. Little change in mRNA abundance of sdDio2 and sdDio3 in the brain was observed during the vitellogenic phases. It is assumed that simultaneous activation and inactivation of THs occur in this area because oral administration of triiodothyronine (T3), but not of thyroxine (T4), upregulated mRNA abundance of both genes in the brain. The transcript levels of sdDio2 in the liver and sdDio3 in the ovary increased as vitellogenesis progressed, suggesting that, through the metabolism of THs, sdDio2 and sdDio3 play a role in vitellogenin synthesis in the liver and yolk accumulation/E2 synthesis in the ovary. Taken together, these results suggest that iodothyronine deiodinases act as a driver for vitellogenesis in tropical damselfish by conversion of THs in certain peripheral tissues.

Daily variation of D2 dopamine receptor transcription in the brain of the Japanese eel Anguilla japonica and its regulation with dopamine and melatonin.

Dopamine plays a crucial role in controlling reproduction in eels, and its action is mediated through D2-type dopamine receptors. D2A and D2B receptors in the Japanese eel Anguilla japonica were cloned and characterized in the present study. Attention (daily expression patterns in the brain and endogenous regulation) was paid to D2B receptor because it is considered to play a crucial role in eel reproduction. The cDNAs of D2A and D2B receptors had open reading frames comprising 456 and 454 amino acid residues, respectively, which were phylogenetically clustered with those of other teleost species. Both receptors were highly expressed in the brain. D2B receptor transcript levels exhibited high day/low night variation in the midbrain and pituitary, suggesting that its transcription in these tissues is regulated in a daily manner, possibly under influence of melatonin. Intraperitoneal injection of dopamine downregulated D2B receptor transcription significantly in the midbrain and moderately in the pituitary within 1 h, but upregulated its transcription in the forebrain. Co-injection of dopamine with its antagonist (domperidone) reversed the effect of dopamine in the pituitary and forebrain, but not in the midbrain, suggesting that the effect of dopamine on D2B receptor transcription differs among brain regions. The same treatment with melatonin resulted in decreased D2B receptor transcription in the midbrain. These findings indicate that dopamine and melatonin have key roles in the daily variation in D2B receptor transcription in the brain of Japanese eel, and that they are related to a daily base secretion of hormones in the hypothalamic-pituitary-gonadal axis in this species.

Verification of differentially expressed genes in relation to hydrostatic pressure in the brain of two wrasse species with high-tide preference in spawning.

Fish that inhabit shallow water are exposed to periodic changes in tidal cues, including hydrostatic pressure (HP). The present study aimed at verifying differentially expressed genes (DEGs) in the brain of the threespot wrasse Halichoeres trimaculatus (tropical species) and the honbera wrasse Halichoeres tenuispinis (temperate species), both of which were exposed to HP at 30 kPa (possible high-tide stimuli in the field) or 1 kPa (low tide) for 3 or 6 h. A de novo assembly yielded 174,710 contigs (63,530 contigs were annotated) from the brain of threespot wrasse. Following RNA sequencing, quantitative PCR confirmed DEGs that were upregulated [AT atypical cadherin 2 (FAT2)] and downregulated [neuronal leucine-rich repeat protein 3 (LRRN3), dual specificity tyrosine phosphorylation-regulated kinase 1 (DYRK), mitogen-activated protein kinase kinase 1 (MAP2K1) and phosphoinositide 3 kinase (PI3K)]. The effect of HP on the transcription of these DEGs (except for MAP2K1) disappeared within 6 h, suggesting that HP is a transitory stimulus occurring at the beginning of the tidal cycle. Similar DEG transcription was observed in the brain of honbera wrasse maintained under HP for 6 h. In situ hybridization of the brain of the threespot wrasse revealed that strong signals of MPA2K1 were seen in the telencephalon, diencephalon and pituitary, whereas those of PI3K were seen in the telencephalon, diencephalon and medulla oblongata. This result suggests that these kinases are involved in sensory function (telencephalon), somatic and visceral function (medullar oblongata) and the neuroendocrine system (diencephalon and pituitary), all of which were related to changes in HP stimuli. Following HP exposure, the transcription of c-fos increased in the pituitary of honbera wrasse, suggesting that external stimuli directly or indirectly activate hormone synthesis at the hypothalamic-pituitary-gonadal axis. It is concluded that HP alters gene expression in relation to neural development and function in the central nervous system and plays a role in exerting tidal-related reproduction and feeding in wrasses.

Annual patterns of ocular melatonin level in the female grass puffer, Takifugu alboplumbeus: possible involvement in seasonal reproductive response.

The aim of this study was to investigate the expression patterns of ocular melatonin in the annual reproductive cycle of the female grass puffer. Spawning season of the female grass puffer is from June to July in Jeju, South Korea. Time-resolved fluoroimmunoassay revealed that levels of ocular melatonin, which show an annual change, peaked in May (spawning season). Additionally, expression of reproductive-related genes also showed annual patterns: GnRH1 peaked in August, GnRH2 peaked in February, GnRH3, Kiss2, and LPXRFa peaked in November. These results suggest that ocular melatonin may be related to the annual reproductive cycle in the grass puffer. To better understand the photic regulation of AANAT1a mRNA in the retina, we observed the nocturnal pattern of ocular melatonin levels daily, which shows a nocturnal pattern in both short photoperiod (SD) and long photoperiod (LD) conditions. In the brain, AANAT2 mRNA also shows a nocturnal pattern in both SD and LD; however, the time of peak expression of AANAT2 mRNA was unchanged in both conditions. Following intraperitoneal injection of melatonin for 2 weeks, expression of GnRH2 and LPXRFa mRNA in the brain significantly increased, while that of Kiss2 mRNA was decreased, suggesting that melatonin has a reproduction-related effect. Furthermore, under SD and LD conditions for 14 weeks, the gonadosomatic index more increased and the maturity of the ovary progressed under LD compared with those under SD, suggesting that the SD photoperiodic signal inactivated ovarian development. These results indicate that the ocular melatonin may have a possible role in the reproductive endocrinology of the grass puffer.

The mRNA expression patterns of kisspeptins, GnRHs, and gonadotropins in the brain and pituitary gland of a tropical damselfish, Chrysiptera cyanea, during the reproductive cycle.

The sapphire devil (Chrysiptera cyanea) is a tropical damselfish that undergoes active reproduction under long-day conditions. To elucidate the physiological regulation of the brain-pituitary-gonadal axis in female sapphire devil, we cloned and characterized the genes of two kisspeptins (kiss1 and kiss2), three gonadotropin-releasing hormones (gnrh1, gnrh2, gnrh3), and the ß-subunit of two gonadotropins (fshß and lhß) and investigated the gene expression changes during ovarian development. Quantitative polymerase chain reaction analyses in various brain parts revealed high expression levels of kiss1, kiss2, and gnrh2 in the diencephalon; gnrh2 and gnrh3 in the telencephalon; and fshß and lhß in the pituitary. In situ hybridization (ISH) analyses revealed positive signals of kiss1 in the dorsal and ventral habenular nucleus and of kiss2 in the dorsal and ventral parts of the nucleus of the lateral recess. This analysis showed gnrh1 expression in the preoptic area (POA), suggesting that GnRH1 plays a stimulating role in the secretion of gonadotropins from the pituitary of the sapphire devil. High transcription levels of kiss1, kiss2, gnrh1, gnrh2, fshß, and lhß were observed in the brain during the late vitellogenic stage, suggesting their involvement in the physiological processes of vitellogenesis. Immersion of fish in estradiol-17ß (E2)-containing seawater resulted in increased expression of kiss2 and gnrh1 in their brains. This study showed that kiss-expressing neurons in the diencephalon are influenced by E2, leading to upregulation of gnrh1 in the POA and of fshß and lhß in the pituitary during vitellogenesis.

Effects of temperature and melatonin on day-night expression patterns of arginine vasotocin and isotocin mRNA in the diencephalon of a temperate wrasse Halichoeres tenuispinis.

Most wrasses are protogynous species that swim to feed, reproduce during the daytime, and bury themselves under the sandy bottom at night. In temperate and subtropical wrasses, low temperature influences emergence from the sandy bottom in the morning, and induces a hibernation-like state in winter. We cloned and characterized the prohormone complementary DNAs (cDNAs) of arginine vasotocin (AVT) and isotocin (IT) in a temperate wrasse (Halichoeres tenuispinis) and examined the effects of day/night and temperature on their expression in the diencephalon, because these neurohypophysial peptides are related to the sex behavior of wrasses. The full-length cDNAs of pro-AVT and pro-IT were 938 base pairs (154 amino acids) and 759 base pairs (156 amino acids) in length, respectively. Both pro-peptides contained a signal sequence followed by the respective hormones and neurophysin connected by a Gly-Lys-Arg bridge. Reverse-transcription polymerase chain reaction (RT-PCR) revealed that pro-AVT mRNA expression was specifically observed in the diencephalon, whereas pro-IT mRNA expression was seen in the whole brain. Quantitative RT-PCR revealed that the mRNA abundance of pro-AVT and pro-IT was higher at midday (zeitgeber time 6; ZT6) than at midnight (ZT18) under 12 h light and 12 h darkness (LD 12:12) conditions, but not under constant light. Intraperitoneal injection of melatonin decreased the mRNA abundance of pro-AVT, but not of pro-IT. When fish were reared under LD 12:12 conditions at 25, 20, and 15 °C, day high and night low mRNA expressions of pro-AVT and pro-IT were maintained. A field survey revealed seasonal variation in the number of swimming fish at observatory sites; many fish emerged from the sandy bottom in summer, but not in winter, suggesting a hibernation-like state under the sandy bottom under low temperature conditions. We conclude that the day-night fluctuation of pro-AVT and pro-IT mRNA abundance in the brain is not affected by temperature and repeated under the sandy bottom in winter.

Molecular cloning of kisspeptin receptor genes (gpr54-1 and gpr54-2) and their expression profiles in the brain of a tropical damselfish during different gonadal stages.

The kisspeptin receptor (GPR54) mediates neuroendocrine control of kisspeptin in the brain and acts as a gateway for a pulsatile release of hypothalamic gonadotropin-releasing hormone. This study aimed to clone two GPR54 genes (gpr54-1 and gpr54-2) from the brain of the sapphire devil Chrysiptera cyanea, a tropical damselfish, and to study their involvement in reproduction. The partial sequences of the sapphire devil gpr54-1 cDNA (1059bp) and gpr54-2 cDNA (1098bp) each had an open reading frame encoding a protein of 353 and 366 amino acids, respectively, both of which had structural features of a G-protein-coupled receptor. The expression of gpr54-1 mRNA was observed in the diencephalon and telencephalon, and gpr54-2 mRNA was found in the optic tectum of sapphire devil. When gpr54-1 and gpr54-2 mRNA levels were examined in the brain of sapphire devil by real-time quantitative polymerase chain reaction (qPCR), they were found to increase during late vitellogenesis and post-spawning. Treatment of fish with estradiol-17ß (Ε2) resulted in an increase in gpr54-1 and gpr54-2 expression in the brain of sapphire devil. Thus, kisspeptin receptors likely mediate the activity of kisspeptin in the brain and are involved in controlling reproductive events in a tropical damselfish.

Existence of a photoinducible phase for ovarian development and photoperiod-related alteration of clock gene expression in a damselfish.

The sapphire devil, Chrysiptera cyanea, is a reef-associated damselfish and their ovarian development can be induced by a long photoperiod. In this study, we demonstrated the existence of a photoinducible phase for the photoperiodic ovarian development in the sapphire devil. Induction of ovarian development under night-interruption light schedules and Nanda-Hamner cycles revealed that the photoinducible phase appeared in a circadian manner between ZT12 and ZT13. To characterize the effect of photoperiod on clock gene expression in the brain of this species, we determined the expression levels of the sdPer1, sdPer2, sdCry1, and sdCry2 clock genes under constant light and dark conditions (LL and DD) and photoperiodic (short and long photoperiods). The expression of sdPer1 exhibited clear circadian oscillation under both LL and DD conditions, while sdPer2 and sdCry1 expression levels were lower under DD than under LL conditions and sdCry2 expression was lower under LL than under DD conditions. These results suggest a key role for sdPer1 in circadian clock cycling and that sdPer2, sdCry1, and sdCry2 are light-responsive clock genes in the sapphire devil. After 1 week under a long photoperiod, we observed photoperiod-related changes in sdPer1, sdPer2, and sdCry2 expression, but not in sdCry1 expression. These results suggest that the expression patterns of some clock genes exhibit seasonal variation according to seasonal changes in day length and that such seasonal alteration of clock gene expression may contribute to seasonal recognition by the sapphire devil.

Effects of different colors of light on melatonin suppression and expression analysis of Aanat1 and melanopsin in the eye of a tropical damselfish.

Ocular melatonin production exhibits a daily rhythm with a decrease during photophase and an increase during scotophase (nocturnal pattern) in teleost fish due to day-night changes in the activity of the rate-limiting melatonin synthesizing enzyme arylalkylamine N-acetyltransferase (AANAT). Acute light exposure during scotophase suppresses AANAT activity and melatonin production in the eyes, suggesting that external light signals are a principal regulator of ocular melatonin synthesis. To better understand the photic regulation of ocular melatonin synthesis in teleost fish, this study sought to characterize the effect of light on ocular melatonin synthesis in the sapphire devil Chrysiptera cyanea, which shows a nocturnal pattern and light-induced inhibition of ocular melatonin production during scotophase. Exposure to three different wavelengths of light (half-peak bandwidth=435-475 nm with a peak of 455 nm, 495-565 nm with a peak of 530 nm, and 607-647 nm with a peak of 627 nm for the blue, green, and red LEDs) for 2h during scotophase resulted in the blue wavelength significantly decreasing ocular melatonin content within 30 min after light exposure. This result clearly indicates that the effective range of visible light on ocular melatonin suppression is distributed within the wavelengths of blue light and that a blue light-sensitive opsin is involved in ocular melatonin suppression in the fish. A PCR-based cloning method revealed the expression of melanopsin, a putative blue light-sensitive nonvisual opsin, in the eyes. Furthermore, in situ hybridization using the sapphire devil Aanat1 and melanopsin RNA probes showed mRNA expressions of both genes in the inner nuclear and ganglion cell layer of the fish retina. These results suggest that melanopsin is a possible candidate photoreceptor involved in ocular melatonin suppression by an external light signal in the sapphire devil.

Effects of dopamine and melatonin treatment on the expression of the genes associated with artificially induced sexual maturation in Japanese eel, Anguilla japonica.

Japanese eel (Anguilla japonica) is a commercially important fish species in Asia. Understanding factors like photoperiod, temperature, and lunar cycles is crucial for successful aquaculture and managing its reproduction. Melatonin and dopamine (DA) are essential for regulating reproduction in vertebrates, including fish. This study investigated the effects of melatonin and DA on the reproductive system of mature male Japanese eels to better understand reproductive regulation in fish. To clarify the effects of these hormones on sexual maturation in eels, a critical stage in the reproductive process, sexual maturation was induced by injecting human chorionic gonadotropin, which stimulates the production of sex hormones. To check the effect of melatonin and DA on sexual maturation, DA, melatonin, and DA + domperidone were intraperitoneally injected into fish from each group (six per treatment) at a dose of 1 mg/kg body weight. The fish were then examined using quantitative RT-PCR by comparing the messenger RNA level of reproduction-related genes (gonadotropin releasing hormone 1; gnrh1, gonadotropin releasing hormone 2; gnrh2, follicle stimulating hormone; fshß, luteinizing hormone; lhß and DA receptor 2b; d2b), involved in the gonadotropic axis in eels, to those that received a control injection. The results indicate significant differences in the expression levels of gnrh1, gnrh2 and d2b in the brain and d2b, fshß, lhß in the pituitary at different stages of sexual maturation. Melatonin appears to enhance the production of sex gonadotropins, whereas DA inhibits them. These findings suggest an interaction between melatonin and DA in regulating reproduction in Japanese eels.

Effects of hydrostatic pressure on monoaminergic activity in the brain of a tropical wrasse, Halicoeres trimaculatus: possible implication for controlling tidal-related reproductive activity.

Most wrasse species in tropical waters exhibit daily spawning synchrony with a preference for high tide. Fish perceive tidal rhythm cues through sensory organs and activate the brain-pituitary-gonadal endocrine axis for synchronous gonadal maturation, although how the tidal-related spawning cycle is controlled endogenously is not known. The purpose of this study was to examine whether hydrostatic pressure has an impact on brain monoamine levels and reproductive activities in the threespot wrasse Halichoeres trimaculatus. The contents of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) in the brain were measured using high-performance liquid chromatography and an electrochemical detection system. Exposing the fish to hydrostatic pressure occurring at a 3-m depth (~30 kPa) resulted in an increase in 5-HIAA/5-HT over 3h and a decrease in DOPAC/DA over 6h. No changes in gonadosomatic index or oocyte diameter were observed between the groups when female fish were reared at 0-m and 3-m depth for 3h. Hydrostatic pressure did not alter pituitary mRNA abundance of follicle stimulating hormone-ß or luteinizing hormone-ß. However, in vitro culture of ovaries from pressurized fish in the presence of human chorionic gonadotropin resulted in an increase in 17α,20ß-dihydroxy-4-pregnen-3-one in the medium. These results suggest that hydrostatic pressure activates oocyte maturation through brain monoaminergic activity in this tropical wrasse species.

Fish sleeping under sandy bottom: interplay of melatonin and clock genes.

Wrasse species exhibit a definite daily rhythm in locomotor activity and bury themselves in the sand at the bottom of the ocean at night. It remains unclear how their behavior in locomotor activity is endogenously regulated. The aim of the present study was to clarify the involvement of melatonin and clock genes (Per1, Per2, Bmal1, and Cry1) in daily and circadian rhythms of the threespot wrasse, Halichoeres trimaculatus, which is a common species in coral reefs. Daily and circadian rhythms in locomotor activity were monitored under conditions of light-dark cycle (LD=12:12), constant light (LL), and darkness (DD). Daily rhythms in locomotor activity were observed under LD and persisted under LL and DD. Melatonin from a cultured pineal gland showed daily variations with an increase during the nighttime and a decrease during daytime, which persisted under DD. Melatonin treatment induced decreases in locomotor activity and respiratory rate, suggesting that melatonin has a sleep-inducing effect. Per1 and Per2 mRNA abundance in the brain under LD showed daily rhythms with an increase around lights on. Robust oscillation of Per1 and Per2 mRNA expression persisted under DD and LL, respectively. Expression of Bmal1 and Cry1 mRNA also showed daily and circadian patterns. These results suggest that clock genes are related to circadian rhythms in locomotor activity and that melatonin plays a role in inducing a sleep-like state after fish bury themselves in the sand. We conclude that the sleep-wake rhythm of the wrasse is regulated by a coordination of melatonin and clock genes.

Anti-Obesity Effects of Polymethoxyflavone-Rich Fraction from Jinkyool (Citrus sunki Hort. ex Tanaka) Leaf on Obese Mice Induced by High-Fat Diet.

Polymethoxyflavones (PMFs) are flavonoids exclusively found in certain citrus fruits and have been reported to be beneficial to human health. Most studies have been conducted with PMFs isolated from citrus peels, while there is no study on PMFs isolated from leaves. In this study, we prepared a PMF-rich fraction (PRF) from the leaves of Citrus sunki Hort ex. Tanaka (Jinkyool) and investigated whether the PRF could improve metabolic decline in obese mice induced by a high-fat diet (HFD) for 5 weeks. The HFD-induced obese mice were assigned into HFD, OR (HFD + orlistat at 15.6 mg/kg of body weight/day), and PRF (HFD + 50, 100, and 200 mg/kg of body weight/day) groups. Orlistat and PRF were orally administered for 5 weeks. At the end of the experiment, the serum biochemical parameters, histology, and gene expression profiles in the tissues of each group were analyzed. The body weight gain of the obese mice was significantly reduced after orlistat and PRF administration for 5 weeks. PRF effectively improved HFD-induced insulin resistance and dyslipidemia. Histological analysis in the liver demonstrated that PRF decreased adipocyte size and potentially improved the liver function, as it inhibited the incidence of fatty liver. PRF activated AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), and hormone-sensitive lipase (HSL) in HFD-induced obese mice. Moreover, liver transcriptome analysis revealed that PRF administration enriched genes mainly related to fatty-acid metabolism and immune responses. Overall, these results suggest that the PRF exerted an anti-obesity effect via the modulation of lipid metabolism.

DNA barcoding and morphological identification of spiny lobsters in South Korean waters: a new record of Panulirus longipes and Panulirus homarus homarus.

To date, 19 species of spiny lobsters from the genus Panulirus have been discovered, of which only P. japonicus, P. penicilatus, P. stimpsoni, and P. versicolor have been documented in South Korean waters. In this study, we aimed to identify and update the current list of spiny lobster species that inhabit South Korean waters based on the morphological features and the phylogenetic profile of cytochrome oxidase I (COI) of mitochondrial DNA (mtDNA). Spiny lobsters were collected from the southern and eastern coasts of Jeju Island, South Korea. Phylogenetic analyses were performed using neighbor-joining (NJ), maximum likelihood (ML), and Bayesian inference (BI) methods. The ML tree was used to determine the spiny lobster lineages, thereby clustering the 17 specimens collected in this study into clades A, B, C, and D, which were reciprocally monophyletic with P. japonicus, P. homarus homarus, P. longipes, and P. stimpsoni, respectively. These clades were also supported by morphological examinations. Interestingly, morphological variations, including the connected pleural and transverse groove at the third abdominal somite, were observed in four specimens that were genetically confirmed as P. japonicus. This finding is novel within the P. japonicus taxonomical reports. Additionally, this study updates the documentation of spiny lobsters inhabiting South Korean waters as P. longipes and P. homarus homarus were recorded for the first time in this region.

Expression of type II iodothyronine deiodinase gene in the brain of a tropical spinefoot, Siganus guttatus.

Type II iodothyronine deiodinase (D2) converts 3,5,3',5'-tetraiodothyronine to 3,5,3'-triiodothyronine and is involved in regulating thyroid hormone-dependent processes in various tissues. D2 mRNA expression in the mediobasal hypothalamus is affected by photoperiod, which influences reproductive processes in temperate birds and mammals. We examined whether D2 mRNA is expressed in the hypothalamus (located in the forebrain within the diencephalon area) and whether its abundance is affected by day length, temperature, or food availability in the tropical spinefoot, Siganus guttatus, which is endemic to tropical monsoon areas. The reverse transcription-polymerase chain reaction (RT-PCR) revealed that D2 mRNA is expressed in various brain regions. The abundance of hypothalamic D2 mRNA was higher at 12.00h than at 06.00h or 24.00h. Rearing fish under constant dark conditions resulted in a decrease in D2 mRNA abundance during the subjective night. A single injection of melatonin lowered D2 mRNA abundance within 3h. Collectively, it appears that hypothalamic D2 mRNA abundance is regulated by the circadian system and/or melatonin. No differences in D2 mRNA abundance were observed, when fish were reared at 20, 25, and 30°C. However, food deprivation stimulated D2 mRNA expression during the daytime. These results suggest that photoperiodic and nutritive conditions affect hypothalamic D2 mRNA expression in S. guttatus.

Molecular cloning and expression of long-wavelength-sensitive cone opsin in the brain of a tropical damselfish.

Ovarian development of the sapphire devil, Chrysiptera cyanea, exhibits photoperiodism and is stimulated under long-day conditions. Previous studies suggest that red light is more effective than green and blue lights for inducing ovarian development. In addition, the extra-retinal photoreception involved in the ovarian development is suggested in this species. The present study aimed to clone the red-light-sensitive cone opsin (sdLWS) of this species, to demonstrate its expression in the brain, and to confirm the effectiveness of red light on the initiation of ovarian development. A phylogenetic analysis revealed that sdLWS belongs to the long-wavelength-sensitive opsin (LWS opsin) group, showing high identity (77-92%) with the LWS opsin of other fishes. RT-PCR showed that sdLWS is expressed in the eye, brain, and ovary. In situ hybridization indicated that sdLWS is expressed in the third ventricle periventricular area in the anterior hypothalamus. Exposing fish to long-day conditions of red light resulted in the appearance of vitellogenic oocytes in the ovary and an increase in the gonadosomatic index within 2weeks, suggesting that fish immediately undergo oocyte development under red light conditions. These results indicate that sdLWS is a possible candidate of deep brain photoreceptor molecule involved in photoperiodic ovarian development in the sapphire devil.

Diurnal expression patterns of neurohypophysial hormone genes in the brain of the threespot wrasse Halichoeres trimaculatus.

The aim of this study was to determine the involvement of neurohypophysial hormones in the diurnal patterns of the threespot wrasse Halichoeres trimaculatus, which is common in coral reefs and exhibits daily behavioral periodicity. Prohormone cDNAs of the neurohypophysial peptides, arginine vasotocin (AVT) and isotocin (IT), were cloned by 3'- and 5'-rapid amplification of cDNA ends (RACE). The distribution and expression patterns of pro-AVT and -IT mRNAs in the brain were determined using reverse-transcription polymerase chain reaction (RT-PCR) and real-time quantitative PCR, respectively. The respective full-length cDNAs of pro-AVT and -IT were 945 and 755 bp in length, respectively. The deduced amino acid sequences for pro-AVT and pro-IT were 154 and 156 residues in length, respectively. Both pro-peptides contained a signal sequence followed by the respective hormones and neurophysin connected by a Gly-Lys-Arg bridge. Pro-AVT mRNA was detected only in the hypothalamus area, while pro-IT mRNA in the whole part of the brain. The relative abundance of pro-AVT and -IT mRNA varied according to time of day; it was significantly greater at 12:00 h than at 24:00 h. Following intraperitoneal administration of melatonin, pro-AVT mRNA abundance in the brain decreased, while pro-IT mRNA abundance remained unchanged. These results demonstrate that daily fluctuations of pro-AVT and pro-IT levels in the brain of threespot wrasse are differentially regulated.