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Circadian rhythm of melatonin secretion and growth-related gene expression in the tiger puffer Takifugu rubripes

Circadian rhythm of melatonin secretion and growth-related gene expression in the tiger puffer... Kim et al. Fisheries and Aquatic Sciences (2017) 20:17 DOI 10.1186/s41240-017-0061-0 RESEARCH ARTICLE Open Access Circadian rhythm of melatonin secretion and growth-related gene expression in the tiger puffer Takifugu rubripes 1† 5† 2 3 4 1,6* Byeong-Hoon Kim , Sung-Pyo Hur , Sang-Woo Hur , Yuki Takeuchi , Akihiro Takemura and Young-Don Lee Abstract: Somatostatin (SS) and growth hormone-releasing hormone (GHRH) are primary factors regulating growth hormone (GH) secretion in the pituitary. To date, it remains unknown how this rhythm is controlled endogenously, although there must be coordination of circadian manners. Melatonin was the main regulator in biological rhythms, and its secretion has fluctuation by photic information. But relationship between melatonin and growth-related genes (ghrh and ss) is unclear. We investigated circadian rhythms of melatonin secretion, ghrh and ss expressions, and correlation between melatonin with growth-related genes in tiger puffer Takifugu rubripes. The melatonin secretion showed nocturnal rhythms under light and dark (LD) conditions. In constant light (LL) condition, melatonin secretion has similar patterns with LD conditions. ss1 mRNA was high during scotophase under LD conditions. But ss1 rhythms disappeared in LL conditions. Ghrh appeared opposite expression compared with melatonin levels or ss1 expression under LD and LL. In the results of the melatonin injection, ghrh and ss1 showed no significant expression compared with control groups. These results suggested that melatonin and growth- related genes have daily or circadian rhythms in the tiger puffer. Further, we need to know mechanisms of each ss and ghrh gene regulation. Keywords: Photoperiod, Melatonin, Somatostatin, GHRH, Tiger puffer Background somatostatin (SS), which are synthesized in the hypo- Biological rhythms are controlled by many environmen- thalamus (Klein and Sheridan 2008; Luque et al. 2008). tal changes including light, temperature, universal gravi- Moreover, IGF-1, which is secreted from the liver, is also tation, and weather conditions (Fraser et al. 1993; known to control body growth through interactivity with Forward et al. 1998; Wan et al. 2013; Guerra-Santos et GH (Wood et al. 2005). Studies show that GH secretion al. 2017). In non-mammalian vertebrates, photic signals is controlled by various environmental factors such as are transmitted via neural pathways from the retina to temperature and photoperiod. the pineal gland. These signals control the secretion of GHRH is a member of the glucagon superfamily, and various hormones (Iigo et al. 1997; Ayson and Takemura its primary function is to stimulate GH synthesis and se- 2006; Revel et al. 2006; Moore and Menaker 2011; Hur cretion by binding to GHRH receptors (GHRHR) in the et al. 2011). anterior pituitary. Recent studies in fish show that Body growth is enhanced by growth hormone (GH), GHRH and pituitary adenylate cyclase-activating peptide which is released from the pituitary gland in vertebrates (PACAP) encodings differ by only a single gene, and GH (McLean et al. 1997; Raven et al. 2012; Fuentes et al. induction in the pituitary gland by GHRH has been 2013). The secretion of GH is primarily controlled by identified (Lee et al. 2007). In contrast, SS has been growth hormone-releasing hormone (GHRH) and widely detected not only in the central nervous system but also in peripheral tissues. It inhibits GH secretion in the pituitary (Very et al. 2001). These two GH- * Correspondence: leemri@jejunu.ac.kr regulating hormones, GHRH and SS, have opposing Equal contributors Marine Science Institute, Jeju National University, Jeju 6333, South Korea functions: GHRH enhances GH release from the pituit- 19-5, Hamdeok 5(o)-gill, Jocheon, Jeju Special Self-Governing Province ary in vivo and in vitro, while SS inhibits its secretion in 695-965, Republic of South Korea many teleost fishes (Canosa et al. 2007). Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kim et al. Fisheries and Aquatic Sciences (2017) 20:17 Page 2 of 8 Melatonin is an indole-derived hormone that is syn- oblongata (Hur et al. 2011) (Fig. 1). Sampling was con- thesized in the retina and pineal gland. The secretion of ducted at 1200 h during the daytime, and sampling this hormone is primarily controlled by environmental methods was explained above. light conditions in vertebrates, including fish. The For the circadian variation, fish were adapted under plasma melatonin level in Mozambique tilapia Oreochro- 12-h light and 12-h dark photoperiod condition mis mossambicus increases during the night and de- (12L:12D, light on = 0800 h and light off = 2000 h) for creases at day (Nikaido et al. 2009). Although it has 1 week. After adapted, fish were divided into two groups; been suggested that melatonin secretions affect physio- 12L:12D group and 24-h light photoperiod condition logical functions, including body growth in fish (Taylor (24L) group. The 12L:12D group fish (n =98, BW et al. 2005; Herrero et al. 2007; De Pedro et al. 2008; 126.2 ± 4.1 g, TL 18.0 ± 0.2 cm) and 24L group fish Maitra et al. 2013), the effect of melatonin on the tran- (n = 98, BW 136.4 ± 3.1 g, TL 19.0 ± 0.2 cm) were scription of growth-related genes (ghrh and ss) is still reared for 3 days. Fish were anesthetized in MS-222 and unclear. killed by decapitation at 4-h intervals for 1 day at zeit- The tiger puffer Takifugu rubripes is a commercially geber time (ZT) 2, ZT 6, ZT 10, ZT 14, ZT18, and ZT valuable species in South Korea. The aim of the present 22 for 12L:12D group and circadian time (CT) 2, CT 6, study is to profile its growth-related gene expressions CT 10, CT 14, CT 18, and CT 22 for 24L group fish. and to evaluate the effect of melatonin on expressions of The two experimental groups were sampled at the same these genes. time in different light conditions. The sampling at dark- ness time was conducted under red dim light, and blood Methods plasma, diencephalon, and pituitary were collected as Animal described above. The collected blood plasma were used A total of 200 tiger puffer T. rubripes (body weight in melatonin levels’ analysis, and diencephalon tissues 128.4 ± 2.1 g, body length 18.7 ± 0.1 cm) were used in were used in ghrh1, ghrh2, and ss mRNA expression ana- this study. Fish were obtained from the Tham-Ra Fishery lysis by fluoroimmuno assay (TR-FIA) and real-time located in Soegwipo, Jeju, South Korea, and transported qPCR. The experimental procedures followed the guid- to Marine Research Institute, Jeju National University. ance approved by the Animal Care and Use committees The fish were acclimatized under natural photoperiod of Jeju National University, Jeju, South Korea. and water temperature (20–21 °C). The fish were fed commercial pellets (Daehan co., MP3, Busan, South Melatonin intraperitoneal injection Korea) equivalent to 1 to 2% of body weight at 0900 and A total of 20 fish were used for the effects of melatonin 1600 h daily. with several growth-related genes. Fish were reared Distribution of growth-related genes in the parts of under natural photoperiod and water temperature. The brain were examined by RT-PCR and real-time quantita- one group of fish was melatonin (Sigma, 1 mg/kg, tive RT-PCR (Real-time PCR). The fish (n = 4) brain n = 10) treated by intraperitoneal injection (i.p.), and the were divided to five portions each, the telencephalon, other group was injected with the only vehicle solution optic tectum, diencephalon, cerebellum, and medulla (0.6% of saline, n = 10) at 1100 h. The fish of melatonin Fig. 1 Diagram showing dorsal view (a) and sagittal plane (b) of the puffer fish brain. Ob olfactory bulb, Te telencephalon, Op optic tectum, Ce cerebellum, P pineal gland, PON preoptic nucleus, Sp spinal cord Kim et al. Fisheries and Aquatic Sciences (2017) 20:17 Page 3 of 8 i.p. and vehicle groups were sampled melatonin i.p. after Table 1 Primer sets used in this study 1 h. Fish were anesthetized with MS-222 before sam- Primers Sequence (5′-3′) GeneBank Accession No. pling, and tissues were collected (diencephalon and pitu- ss1- F TCTGCT ACT GCAGT CCAACG CCTT XM_003968318 itary) by decapitation. The collected samples were kept ssl-R CAGCCAGAGCAAGGTT at −80 °C until the analysis. ghrh 1- T ACAGCGT CAT CT GCT CACC DQ659331 Plasma melatonin measurement ghrh 1- GAAGGAGT AGAGGT CGT CAGC The levels of melatonin were measured by time-resolved fluoroimmuno assay (TR-FIA) according to the previous re- ghrhl-V AGCGT CTT CT GCACACCT AT C DQ659332 port (Takemura et al. 2004). Briefly, a 96-well plate (AGC ghrh 1- AGT ACGCAT CGTCGTCTACC Techno Glass) was coated with 50 mM carbonate buffer, pH 9.6 (100 ll/well), containing a melatonin-bovine serum yS- GCCAT CCTT CCTT GGT AT GGA U37499 albumin (BSA) conjugate (5 ng/ml) for 2 h at 24 ± 0.5 °C in actin-F an incubator (Sanyo). After three washes with DELFIA yS- GT CGT ACT CCT GCTT GCT GA wash buffer, 50 ll of samples/standards (7.8–4000 pg/ml) actin-R and 50 ll of anti-melatonin (1:200,000 in assay buffer) were placed in the wells. The plate was incubated overnight at 4 °C. After washing, 100 ll of the secondary antibody 1 min. Growth-related gene expressions in diencephalon were against rabbit immunoglobulin G labeled with europium normalized to amount of the internal control β-actin gene. (1:1000 in assay buffer) was added to the well and incu- bated at 24 °C for 1 h. After washing, 50 ll of DELFIA en- Statistical analysis hancement solution was added to the wells. The signal in The plasma melatonin levels and growth-related genes each well was quantified using a time-resolved fluorometer were expressed as means ± SEM and considered signifi- (Arcus1234, Wallac, Oy, Finland). The composition of the cantly different at P < 0.05. Data were analyzed by one- assay buffer (pH 7.75) used in the present study was way analysis of variance (ANOVA) followed by Tukey’s 0.05 M Tris, 0.9% NaCl, 0.5% BSA, (Sigma), 0.05% NaN3, test to assess statistically significant differences among the 0.01% Tween 40, and 20 lM diethylenetriaminepentaacetic different time points in the daily and circadian variation acid (DTPA, Kanto Chemicals, Tokyo, Japan). DELFIA experiments. The significant differences of growth-related washing buffer (pH 7.75) contained 0.05 M Tris, 0.9% gene expression after melatonin injection were tested by NaCl, 0.1% NaN3, and 0.01% Tween 20. Student’s t test. P < 0.05 was considered significant. RT-PCR and real-time quantitative PCR Results Total RNA was extracted from the tissues of tiger puffer The mRNA expression of growth-related genes in central using the RNAiso Reagent (TaKaRa Bio, Japan) after abso- nerves tissues lutely homogenizing the samples. And 1 μg of total RNA We profiled the distribution of the growth-related gene was used for synthesis of first strand cDNA by Transcrip- expression in the several parts of the tiger puffer brain tor First strand cDNA synthesis kit (Roche Diagnostics, by RT-PCR (Fig. 2a) and real-time qPCR (Fig. 2b–d). Mannheim, Germany). Primer sets of each genes were de- The ss1 expression was detected in the telencephalon signed by isolated ghrh1 (Gene bank, accession number; (Te), optic tectum (Op), diencephalon (Di), and medulla DQ659331), ghrh2 (DQ659332), ss1 (XM_003968318), oblongata (ME) but not in the cerebellum (Ce), while and β-actin (U37499) of tiger puffer from National Center the ghrh1 and ghrh2 seemed to express in the all parts of for Biotechnology Information (NCBI, Table 1). For the the brain (Fig. 2a). RT-PCR, each PCR reaction mix contained 50% of Emer- The ss1 was significantly highly expressed in the Te, Di, ald PCR Master mix (TaKaRa-Bio), 10 μmofeach forward and MEthanOpand Ce (Fig.2b).The significantlyhighest and reverse primer, and 50 ng of cDNA template. The RT- expression of ghrh1 was observed in the ME, and the high- PCR cycling conditions were followed by 28 cycles of de- est expression of ghrh2 was detected in the Te (Fig. 2c, d). naturation for 45 s at 94 °C, annealing for 45 s at 58 °C, and extension for 1 min at 72 °C. The real-time quantita- Daily and circadian rhythm of melatonin and growth- tive PCR was performed using the CFX™ Real-time System related genes (Bio-Rad, Hercules, CA, USA) with 20 ng of cDNA using Plasma melatonin levels were significantly elevated at the FastStart Universal SYBR Green Master (Roche Diagnos- middle of the night (ZT18), and its higher level was kept tics). The real-time quantitative PCR amplification was until the dark phase under 12L:12D conditions (Fig. 3a). performed by initial denaturation at 95 °C for 10 min, 40 cy- When the fish were reared under 24L conditions, the cles of 95 °C for15s,60°Cfor 1 min,and last 60 °C for plasma melatonin showed similar patterns with that under Kim et al. Fisheries and Aquatic Sciences (2017) 20:17 Page 4 of 8 Fig. 2 Tissue-specific expression of growth-related genes in the portion of the brain of tiger puffer. The brain was further divided into five parts. They were analyzed by RT-PCR (a) and qPCR (b–d). The expression of β-actin mRNA was used as reference. Te telencephalon, Op optic tectum, Di diencephalon, Ce cerebellum, Me medulla oblongata, N.C negative control, M 100 bp DNA ladder marker. Means represented by different letters are significant (P < 0.05). Values are mean ± SEM 12L:12D conditions (Fig. 3b). Under 12L:12D conditions, ss1 Discussion and conclusions expression in the diencephalon was more increased during Photoperiod is a well-known regulation factor for many scotophase than photophase (Fig. 4a). ss1 mRNA showed physiological responses in fish, including growth and de- the highest expression at ZT18, and the lowest expression velopment (Norberg et al. 2001; Taylor et al. 2005; Biswas was detected at ZT2. However, this significant (P <0.05) ex- et al. 2008; Gunnarsson et al. 2012). In this study, we de- pression patterns disappear under 24L conditions (Fig. 4b). cided to evaluate the effect of light/dark cycle on the ex- Each ghrh1 and ghrh2 mRNA in the diencephalon pression of growth-related genes in the tiger puffer. showed alike expression patterns under 12L:12D and The distribution of growth-related genes in the brain 24L conditions. Expression of ghrh1 mRNA was more tissue was evaluated using RT-PCR and qPCR. The data increased during photophase than scotophase under revealed widespread distribution of these genes in the 12L:12D conditions, but its rapidly low expression was brain (Fig. 2), similar to that in the orange-spotted detected in ZT6 (Fig. 5a). Under 24L conditions, ghrh1 grouper Epinephelus coioides (Xing et al. 2005; Qian et mRNA showed different expression patterns compared al. 2012). In the orange-spotted grouper, the expression with 12L:12D conditions, and this mRNA detected no of ss mRNA was detected in all brain regions, excluding sudden low expression in CT6 (Fig. 5b). ghrh2 mRNA the epithalamus. Ghrh mRNA was also expressed in showed alike expression pattern with ghrh1 mRNA many brain regions. These data suggest that ss and ghrh under all experiment conditions (Fig. 6a, b). This gene expression may mediate not only GH secretion in the pi- revealed significant expressions under 12L:12D and 24 L tuitary but also multiple biological functions in the tiger conditions. But, this gene detected no significant expres- puffer. sion between photophase and scotophase. The daily and circadian regulation of melatonin is well known in vertebrates, including fish. Plasma melatonin levels in the Senegalese sole Solea senegalensis showed Effect of melatonin treatment on the expressions of typical daily rhythms consisting of low levels during growth-related genes photophase and high levels during scotophase (Bayarri The expression of growth-related genes in the dienceph- et al. 2004). For tench Tinca tinca identified the circa- alon and pituitary after melatonin i.p. were analyzed by dian secretion of melatonin under continuous dark con- qPCR. One hour after melatonin i.p., ss1 mRNA showed ditions as well as light and dark conditions (Oliveira et higher expression levels in melatonin group than saline al. 2009). Our experimental data showed similar results; group (Fig. 7a). However, ghrh1 and ghrh2 mRNA after plasma melatonin secretion in tiger puffers increased melatonin i.p. showed no different expression in the during the dark phase in 12L:12D conditions (Fig. 3a). melatonin group with saline group (Fig. 7b, c). Similar melatonin secretion patterns were found during Kim et al. Fisheries and Aquatic Sciences (2017) 20:17 Page 5 of 8 Fig. 4 Daily and circadian expressions of ss1 mRNA in the Fig. 3 Daily and circadian rhythms of melatonin secretion in the diencephalon of tiger puffer under 12L:12D (a) and continuous light plasma of tiger puffer under 12L:12D (a) and continuous light (b) (b) conditions. The relative values of somatostatin1 mRNA conditions. Means represented by different letters are significant expressions were normalized against β-actin and then averaged. (P < 0.05). Values are mean ± SEM Means represented by different letters are significant (P < 0.05). Values are mean ± SEM continuous light conditions (Fig. 3b). In some fishes, the rise of melatonin secretion during scotophase is con- during photophase (Peinado et al. 1990; Ishikawa et al. trolled using an endogenous clock. In pike Esox and zeb- 1997). We investigated biological rhythms of growth- rafish Danio rerio, the expression of the melatonin related genes in the diencephalon of the tiger puffer. synthesis gene (arylalkylamine N-acetyltransferase The expression patterns of ss1 mRNA were similar to (AANAT)) shows nocturnal rhythms in 12L:12D condi- those shown in previous studies. The ss1 mRNA expres- tions, and this gene has similar expression rhythms dur- sion in the diencephalon of tiger puffers increased dur- ing continuous light or dark conditions (Bégay et al. ing the dark phase in 12L:12D conditions (Fig. 4). 1998). These fish have circadian rhythms of high secre- However, ss1 mRNA expression rhythm disappeared tion during scotophase caused by the circadian oscilla- under 24L conditions. Therefore, ss1mRNA suggest to tion of the AANAT gene via an endogenous clock. have the daily rhythm according to light and dark. Therefore, we suggest that melatonin have the circadian In our presents, ghrh1 and ghrh2 mRNA exhibited diur- rhythm in the tiger puffer. nal rhythms in tiger puffers (Figs. 5 and 6). In rats, ghrh SS and GHRH regulate the synthesis and secretion of mRNA expression also exhibited diurnal rhythms (Bredow GH in the pituitary of vertebrates, including fish et al. 1996). Peak expression of hypothalamic ghrh mRNA (Bertherat et al. 1995; Sheridan and Hagemeister 2010). occurred during the early part of photophase in rats. The However, SS and GHRH rhythms in fish are not well ghrh expression gradually decreased up to scotophase, and understood since the majority of research has focused the mRNA expressions maintained low levels during sco- on mammals. Circadian variation of SS levels in rat tophase. Diurnal rhythms of hypothalamic ghrh mRNA plasma showed more elevation during scotophase than were also observed in rats (Gardi et al. 1999). In these Kim et al. Fisheries and Aquatic Sciences (2017) 20:17 Page 6 of 8 Fig. 5 Daily and circadian expressions of ghrh1 mRNA in the Fig. 6 Daily and circadian expressions of ghrh2 mRNA in the diencephalon of tiger puffer under 12L:12D (a) and continuous light diencephalon of tiger puffer under 12L:12D (a) and continuous light (b) conditions. The relative values of ghrh1 mRNA expressions were (b) conditions. The relative values of ghrh2 mRNA expressions were normalized against β-actin and then averaged. Means represented normalized against β-actin and then averaged. Means represented by different letters are significant (P < 0.05). Values are mean ± SEM by different letters are significant (P < 0.05). Values are mean ± SEM studies, hypothalamic ghrh levels increased rapidly in the expressions in tiger puffers. In our experiment, ss1 mRNA first hour after light onset, then decreased for 4 h after in diencephalon of tiger puffer was showed inducing expres- light onset. The ghrh level gradually increased up to sion by melatonini.p (Fig.7a).But,noeffectof melatonin the beginning of scotophase and then steadily de- on mRNA expressions of ghrh1 and ghrh2 was detected creased during scotophase. The ghrh1 and ghrh2 (Fig. 7b, c). This suggests that the daily fluctuation of ss1 mRNA rhythms in tiger puffers in this study were gene in the tiger puffer is controlled by melatonin, but not similar to the ghrh mRNA rhythms in rats. The ex- ghrh gene. Few studies have addressed the correlation of pression of ghrh1 mRNA in tiger puffers was high in melatonin with SS or GHRH in vertebrates, including mam- the early part of photophase and then decreased to- mals. Our review of the literature found studies only on the ward the midpoint of photophase. Further, the ghrh1 relationship of melatonin to SS in the rat hippocampus mRNA increased from the midpoint up to the end of (Izquierdo-Claros et al. 2004). This study showed a reduc- photophase. During scotophase, ghrh1 mRNA showed tion in SS levels through decreasing somatostatinergic sys- prolonged low expression levels. Therefore, we sug- tem activity, including inhibition of SS receptor activity by gest that tiger puffers have daily rhythms of ghrh ex- melatonin in the hippocampus. Although this finding seems pression that vary with light and dark conditions. to conflict with our results, we suggest that daily and circa- Our results showed that the ss1 mRNA expression pattern dian rhythms of growth-related genes are directly or indir- was similar to the pattern of melatonin secretion, but ghrh1 ectly controlled by melatonin in the tiger puffer. and ghrh2 mRNA expression patterns were opposite to Briefly, our results suggest that melatonin secretion and those of melatonin secretion. We then examined the correl- growth-related gene expressions follow daily and circadian ation of melatonin secretion and growth-related gene rhythms in the tiger puffer. As a result, melatonin showed Kim et al. Fisheries and Aquatic Sciences (2017) 20:17 Page 7 of 8 Acknowledgements This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2012R1A6A3A04041089). Funding This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF). Availability of data and materials Not applicable. Authors’ contributions BH and SP designed and carried out the data analysis and manuscript writing. SW participated in the fish sampling and data analysis. YT and AT participated in the plasma melatonin data analysis. YD participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. Ethics approval All experiments were conducted in compliance with both the Animal Care and Use Committee guidelines of the JeJu National University. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author details Marine Science Institute, Jeju National University, Jeju 6333, South Korea. Aquafeed Research Center, National Institute of Fisheries Science (NIFS), Pohang 37517, South Korea. Department of Electrical Engineering and Bioscience, School of Advanced Science and Engineering, Waseda University, Fig. 7 Expression of growth-related genes mRNA in the tiger puffer Shinjuku, Tokyo 162-8480, Japan. Department of Chemistry, Biology and diencephalon by after 1 h of melatonin i.p. Means represented by Marine Sciences, Faculty of Science, University of the Ryukyus, Nishihara, different letters are significant (P < 0.05). The relative values of (a) Okinawa 903-0213, Japan. Jeju International Marine Science Research & ss1, (b) ghrh1 and (c) ghrh2 mRNA expressions were normalized Logistics Center, Korea Institute of Ocean Science & Technology, Jeju 63349, South Korea. 19-5, Hamdeok 5(o)-gill, Jocheon, Jeju Special Self-Governing against β-actin and then averaged. Values are mean ± SEM Province 695-965, Republic of South Korea. Received: 12 February 2017 Accepted: 20 July 2017 that nocturnal rhythm in 12L:12D condition and this rhythm was similarly showed in 24L condition compared with 12L:12D condition. Through these results, melatonin References Ayson FG, Takemura A. Daily expression patterns for mRNAs of GH, PRL, SL, IGF-I is suggested to have the circadian rhythm regardless of the and IGF-II in juvenile rabbitfish, Siganus guttatus, during 24-h light and dark day and night. The ss1 expression appears to be induction cycles. Gen Comp Endocrinol. 2006;149:261–8. by melatonin treatment. However, ss1 expression rhythm Bayarri MJ, Munoz-Cueto JA, López-Olmeda JF, Vera LM, De Lama MR, Madrid JA, Sanchez-Vazquez FJ. Daily locomotor activity and melatonin rhythms in showed different expression rhythm when compared with Senegal sole (Solea senegalensis). Physiol Behav. 2004;81:577–83. melatonin rhythm under 24L condition. 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Photic and circadian regulation of melatonin and we will help you at every step: production in the Mozambique tilapia Oreochromis mossambicus. Comp • We accept pre-submission inquiries Biochem Physiol A Mol Integr Physiol. 2009;152:77–82. Norberg B, Weltzien FA, Karlsen Ø, Holm JC. Effects of photoperiod on � Our selector tool helps you to find the most relevant journal sexual maturation and somatic growth in male Atlantic halibut � We provide round the clock customer support (Hippoglossus hippoglossus L.). Comp Biochem Physiol B Biochem Mol � Convenient online submission Biol. 2001;129:357–65. Oliveira C, Garcia EM, López-Olmeda JF, Sánchez-Vázquez FJ. Daily and circadian � Thorough peer review melatonin release in vitro by the pineal organ of two nocturnal teleost � Inclusion in PubMed and all major indexing services species: Senegal sole (Solea senegalensis) and tench (Tinca tinca). Comp � Maximum visibility for your research Biochem Physiol A Mol Integr Physiol. 2009;153:297–302. 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Circadian rhythm of melatonin secretion and growth-related gene expression in the tiger puffer Takifugu rubripes

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Life Sciences; Fish & Wildlife Biology & Management; Marine & Freshwater Sciences; Zoology; Animal Ecology
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Abstract

Kim et al. Fisheries and Aquatic Sciences (2017) 20:17 DOI 10.1186/s41240-017-0061-0 RESEARCH ARTICLE Open Access Circadian rhythm of melatonin secretion and growth-related gene expression in the tiger puffer Takifugu rubripes 1† 5† 2 3 4 1,6* Byeong-Hoon Kim , Sung-Pyo Hur , Sang-Woo Hur , Yuki Takeuchi , Akihiro Takemura and Young-Don Lee Abstract: Somatostatin (SS) and growth hormone-releasing hormone (GHRH) are primary factors regulating growth hormone (GH) secretion in the pituitary. To date, it remains unknown how this rhythm is controlled endogenously, although there must be coordination of circadian manners. Melatonin was the main regulator in biological rhythms, and its secretion has fluctuation by photic information. But relationship between melatonin and growth-related genes (ghrh and ss) is unclear. We investigated circadian rhythms of melatonin secretion, ghrh and ss expressions, and correlation between melatonin with growth-related genes in tiger puffer Takifugu rubripes. The melatonin secretion showed nocturnal rhythms under light and dark (LD) conditions. In constant light (LL) condition, melatonin secretion has similar patterns with LD conditions. ss1 mRNA was high during scotophase under LD conditions. But ss1 rhythms disappeared in LL conditions. Ghrh appeared opposite expression compared with melatonin levels or ss1 expression under LD and LL. In the results of the melatonin injection, ghrh and ss1 showed no significant expression compared with control groups. These results suggested that melatonin and growth- related genes have daily or circadian rhythms in the tiger puffer. Further, we need to know mechanisms of each ss and ghrh gene regulation. Keywords: Photoperiod, Melatonin, Somatostatin, GHRH, Tiger puffer Background somatostatin (SS), which are synthesized in the hypo- Biological rhythms are controlled by many environmen- thalamus (Klein and Sheridan 2008; Luque et al. 2008). tal changes including light, temperature, universal gravi- Moreover, IGF-1, which is secreted from the liver, is also tation, and weather conditions (Fraser et al. 1993; known to control body growth through interactivity with Forward et al. 1998; Wan et al. 2013; Guerra-Santos et GH (Wood et al. 2005). Studies show that GH secretion al. 2017). In non-mammalian vertebrates, photic signals is controlled by various environmental factors such as are transmitted via neural pathways from the retina to temperature and photoperiod. the pineal gland. These signals control the secretion of GHRH is a member of the glucagon superfamily, and various hormones (Iigo et al. 1997; Ayson and Takemura its primary function is to stimulate GH synthesis and se- 2006; Revel et al. 2006; Moore and Menaker 2011; Hur cretion by binding to GHRH receptors (GHRHR) in the et al. 2011). anterior pituitary. Recent studies in fish show that Body growth is enhanced by growth hormone (GH), GHRH and pituitary adenylate cyclase-activating peptide which is released from the pituitary gland in vertebrates (PACAP) encodings differ by only a single gene, and GH (McLean et al. 1997; Raven et al. 2012; Fuentes et al. induction in the pituitary gland by GHRH has been 2013). The secretion of GH is primarily controlled by identified (Lee et al. 2007). In contrast, SS has been growth hormone-releasing hormone (GHRH) and widely detected not only in the central nervous system but also in peripheral tissues. It inhibits GH secretion in the pituitary (Very et al. 2001). These two GH- * Correspondence: leemri@jejunu.ac.kr regulating hormones, GHRH and SS, have opposing Equal contributors Marine Science Institute, Jeju National University, Jeju 6333, South Korea functions: GHRH enhances GH release from the pituit- 19-5, Hamdeok 5(o)-gill, Jocheon, Jeju Special Self-Governing Province ary in vivo and in vitro, while SS inhibits its secretion in 695-965, Republic of South Korea many teleost fishes (Canosa et al. 2007). Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kim et al. Fisheries and Aquatic Sciences (2017) 20:17 Page 2 of 8 Melatonin is an indole-derived hormone that is syn- oblongata (Hur et al. 2011) (Fig. 1). Sampling was con- thesized in the retina and pineal gland. The secretion of ducted at 1200 h during the daytime, and sampling this hormone is primarily controlled by environmental methods was explained above. light conditions in vertebrates, including fish. The For the circadian variation, fish were adapted under plasma melatonin level in Mozambique tilapia Oreochro- 12-h light and 12-h dark photoperiod condition mis mossambicus increases during the night and de- (12L:12D, light on = 0800 h and light off = 2000 h) for creases at day (Nikaido et al. 2009). Although it has 1 week. After adapted, fish were divided into two groups; been suggested that melatonin secretions affect physio- 12L:12D group and 24-h light photoperiod condition logical functions, including body growth in fish (Taylor (24L) group. The 12L:12D group fish (n =98, BW et al. 2005; Herrero et al. 2007; De Pedro et al. 2008; 126.2 ± 4.1 g, TL 18.0 ± 0.2 cm) and 24L group fish Maitra et al. 2013), the effect of melatonin on the tran- (n = 98, BW 136.4 ± 3.1 g, TL 19.0 ± 0.2 cm) were scription of growth-related genes (ghrh and ss) is still reared for 3 days. Fish were anesthetized in MS-222 and unclear. killed by decapitation at 4-h intervals for 1 day at zeit- The tiger puffer Takifugu rubripes is a commercially geber time (ZT) 2, ZT 6, ZT 10, ZT 14, ZT18, and ZT valuable species in South Korea. The aim of the present 22 for 12L:12D group and circadian time (CT) 2, CT 6, study is to profile its growth-related gene expressions CT 10, CT 14, CT 18, and CT 22 for 24L group fish. and to evaluate the effect of melatonin on expressions of The two experimental groups were sampled at the same these genes. time in different light conditions. The sampling at dark- ness time was conducted under red dim light, and blood Methods plasma, diencephalon, and pituitary were collected as Animal described above. The collected blood plasma were used A total of 200 tiger puffer T. rubripes (body weight in melatonin levels’ analysis, and diencephalon tissues 128.4 ± 2.1 g, body length 18.7 ± 0.1 cm) were used in were used in ghrh1, ghrh2, and ss mRNA expression ana- this study. Fish were obtained from the Tham-Ra Fishery lysis by fluoroimmuno assay (TR-FIA) and real-time located in Soegwipo, Jeju, South Korea, and transported qPCR. The experimental procedures followed the guid- to Marine Research Institute, Jeju National University. ance approved by the Animal Care and Use committees The fish were acclimatized under natural photoperiod of Jeju National University, Jeju, South Korea. and water temperature (20–21 °C). The fish were fed commercial pellets (Daehan co., MP3, Busan, South Melatonin intraperitoneal injection Korea) equivalent to 1 to 2% of body weight at 0900 and A total of 20 fish were used for the effects of melatonin 1600 h daily. with several growth-related genes. Fish were reared Distribution of growth-related genes in the parts of under natural photoperiod and water temperature. The brain were examined by RT-PCR and real-time quantita- one group of fish was melatonin (Sigma, 1 mg/kg, tive RT-PCR (Real-time PCR). The fish (n = 4) brain n = 10) treated by intraperitoneal injection (i.p.), and the were divided to five portions each, the telencephalon, other group was injected with the only vehicle solution optic tectum, diencephalon, cerebellum, and medulla (0.6% of saline, n = 10) at 1100 h. The fish of melatonin Fig. 1 Diagram showing dorsal view (a) and sagittal plane (b) of the puffer fish brain. Ob olfactory bulb, Te telencephalon, Op optic tectum, Ce cerebellum, P pineal gland, PON preoptic nucleus, Sp spinal cord Kim et al. Fisheries and Aquatic Sciences (2017) 20:17 Page 3 of 8 i.p. and vehicle groups were sampled melatonin i.p. after Table 1 Primer sets used in this study 1 h. Fish were anesthetized with MS-222 before sam- Primers Sequence (5′-3′) GeneBank Accession No. pling, and tissues were collected (diencephalon and pitu- ss1- F TCTGCT ACT GCAGT CCAACG CCTT XM_003968318 itary) by decapitation. The collected samples were kept ssl-R CAGCCAGAGCAAGGTT at −80 °C until the analysis. ghrh 1- T ACAGCGT CAT CT GCT CACC DQ659331 Plasma melatonin measurement ghrh 1- GAAGGAGT AGAGGT CGT CAGC The levels of melatonin were measured by time-resolved fluoroimmuno assay (TR-FIA) according to the previous re- ghrhl-V AGCGT CTT CT GCACACCT AT C DQ659332 port (Takemura et al. 2004). Briefly, a 96-well plate (AGC ghrh 1- AGT ACGCAT CGTCGTCTACC Techno Glass) was coated with 50 mM carbonate buffer, pH 9.6 (100 ll/well), containing a melatonin-bovine serum yS- GCCAT CCTT CCTT GGT AT GGA U37499 albumin (BSA) conjugate (5 ng/ml) for 2 h at 24 ± 0.5 °C in actin-F an incubator (Sanyo). After three washes with DELFIA yS- GT CGT ACT CCT GCTT GCT GA wash buffer, 50 ll of samples/standards (7.8–4000 pg/ml) actin-R and 50 ll of anti-melatonin (1:200,000 in assay buffer) were placed in the wells. The plate was incubated overnight at 4 °C. After washing, 100 ll of the secondary antibody 1 min. Growth-related gene expressions in diencephalon were against rabbit immunoglobulin G labeled with europium normalized to amount of the internal control β-actin gene. (1:1000 in assay buffer) was added to the well and incu- bated at 24 °C for 1 h. After washing, 50 ll of DELFIA en- Statistical analysis hancement solution was added to the wells. The signal in The plasma melatonin levels and growth-related genes each well was quantified using a time-resolved fluorometer were expressed as means ± SEM and considered signifi- (Arcus1234, Wallac, Oy, Finland). The composition of the cantly different at P < 0.05. Data were analyzed by one- assay buffer (pH 7.75) used in the present study was way analysis of variance (ANOVA) followed by Tukey’s 0.05 M Tris, 0.9% NaCl, 0.5% BSA, (Sigma), 0.05% NaN3, test to assess statistically significant differences among the 0.01% Tween 40, and 20 lM diethylenetriaminepentaacetic different time points in the daily and circadian variation acid (DTPA, Kanto Chemicals, Tokyo, Japan). DELFIA experiments. The significant differences of growth-related washing buffer (pH 7.75) contained 0.05 M Tris, 0.9% gene expression after melatonin injection were tested by NaCl, 0.1% NaN3, and 0.01% Tween 20. Student’s t test. P < 0.05 was considered significant. RT-PCR and real-time quantitative PCR Results Total RNA was extracted from the tissues of tiger puffer The mRNA expression of growth-related genes in central using the RNAiso Reagent (TaKaRa Bio, Japan) after abso- nerves tissues lutely homogenizing the samples. And 1 μg of total RNA We profiled the distribution of the growth-related gene was used for synthesis of first strand cDNA by Transcrip- expression in the several parts of the tiger puffer brain tor First strand cDNA synthesis kit (Roche Diagnostics, by RT-PCR (Fig. 2a) and real-time qPCR (Fig. 2b–d). Mannheim, Germany). Primer sets of each genes were de- The ss1 expression was detected in the telencephalon signed by isolated ghrh1 (Gene bank, accession number; (Te), optic tectum (Op), diencephalon (Di), and medulla DQ659331), ghrh2 (DQ659332), ss1 (XM_003968318), oblongata (ME) but not in the cerebellum (Ce), while and β-actin (U37499) of tiger puffer from National Center the ghrh1 and ghrh2 seemed to express in the all parts of for Biotechnology Information (NCBI, Table 1). For the the brain (Fig. 2a). RT-PCR, each PCR reaction mix contained 50% of Emer- The ss1 was significantly highly expressed in the Te, Di, ald PCR Master mix (TaKaRa-Bio), 10 μmofeach forward and MEthanOpand Ce (Fig.2b).The significantlyhighest and reverse primer, and 50 ng of cDNA template. The RT- expression of ghrh1 was observed in the ME, and the high- PCR cycling conditions were followed by 28 cycles of de- est expression of ghrh2 was detected in the Te (Fig. 2c, d). naturation for 45 s at 94 °C, annealing for 45 s at 58 °C, and extension for 1 min at 72 °C. The real-time quantita- Daily and circadian rhythm of melatonin and growth- tive PCR was performed using the CFX™ Real-time System related genes (Bio-Rad, Hercules, CA, USA) with 20 ng of cDNA using Plasma melatonin levels were significantly elevated at the FastStart Universal SYBR Green Master (Roche Diagnos- middle of the night (ZT18), and its higher level was kept tics). The real-time quantitative PCR amplification was until the dark phase under 12L:12D conditions (Fig. 3a). performed by initial denaturation at 95 °C for 10 min, 40 cy- When the fish were reared under 24L conditions, the cles of 95 °C for15s,60°Cfor 1 min,and last 60 °C for plasma melatonin showed similar patterns with that under Kim et al. Fisheries and Aquatic Sciences (2017) 20:17 Page 4 of 8 Fig. 2 Tissue-specific expression of growth-related genes in the portion of the brain of tiger puffer. The brain was further divided into five parts. They were analyzed by RT-PCR (a) and qPCR (b–d). The expression of β-actin mRNA was used as reference. Te telencephalon, Op optic tectum, Di diencephalon, Ce cerebellum, Me medulla oblongata, N.C negative control, M 100 bp DNA ladder marker. Means represented by different letters are significant (P < 0.05). Values are mean ± SEM 12L:12D conditions (Fig. 3b). Under 12L:12D conditions, ss1 Discussion and conclusions expression in the diencephalon was more increased during Photoperiod is a well-known regulation factor for many scotophase than photophase (Fig. 4a). ss1 mRNA showed physiological responses in fish, including growth and de- the highest expression at ZT18, and the lowest expression velopment (Norberg et al. 2001; Taylor et al. 2005; Biswas was detected at ZT2. However, this significant (P <0.05) ex- et al. 2008; Gunnarsson et al. 2012). In this study, we de- pression patterns disappear under 24L conditions (Fig. 4b). cided to evaluate the effect of light/dark cycle on the ex- Each ghrh1 and ghrh2 mRNA in the diencephalon pression of growth-related genes in the tiger puffer. showed alike expression patterns under 12L:12D and The distribution of growth-related genes in the brain 24L conditions. Expression of ghrh1 mRNA was more tissue was evaluated using RT-PCR and qPCR. The data increased during photophase than scotophase under revealed widespread distribution of these genes in the 12L:12D conditions, but its rapidly low expression was brain (Fig. 2), similar to that in the orange-spotted detected in ZT6 (Fig. 5a). Under 24L conditions, ghrh1 grouper Epinephelus coioides (Xing et al. 2005; Qian et mRNA showed different expression patterns compared al. 2012). In the orange-spotted grouper, the expression with 12L:12D conditions, and this mRNA detected no of ss mRNA was detected in all brain regions, excluding sudden low expression in CT6 (Fig. 5b). ghrh2 mRNA the epithalamus. Ghrh mRNA was also expressed in showed alike expression pattern with ghrh1 mRNA many brain regions. These data suggest that ss and ghrh under all experiment conditions (Fig. 6a, b). This gene expression may mediate not only GH secretion in the pi- revealed significant expressions under 12L:12D and 24 L tuitary but also multiple biological functions in the tiger conditions. But, this gene detected no significant expres- puffer. sion between photophase and scotophase. The daily and circadian regulation of melatonin is well known in vertebrates, including fish. Plasma melatonin levels in the Senegalese sole Solea senegalensis showed Effect of melatonin treatment on the expressions of typical daily rhythms consisting of low levels during growth-related genes photophase and high levels during scotophase (Bayarri The expression of growth-related genes in the dienceph- et al. 2004). For tench Tinca tinca identified the circa- alon and pituitary after melatonin i.p. were analyzed by dian secretion of melatonin under continuous dark con- qPCR. One hour after melatonin i.p., ss1 mRNA showed ditions as well as light and dark conditions (Oliveira et higher expression levels in melatonin group than saline al. 2009). Our experimental data showed similar results; group (Fig. 7a). However, ghrh1 and ghrh2 mRNA after plasma melatonin secretion in tiger puffers increased melatonin i.p. showed no different expression in the during the dark phase in 12L:12D conditions (Fig. 3a). melatonin group with saline group (Fig. 7b, c). Similar melatonin secretion patterns were found during Kim et al. Fisheries and Aquatic Sciences (2017) 20:17 Page 5 of 8 Fig. 4 Daily and circadian expressions of ss1 mRNA in the Fig. 3 Daily and circadian rhythms of melatonin secretion in the diencephalon of tiger puffer under 12L:12D (a) and continuous light plasma of tiger puffer under 12L:12D (a) and continuous light (b) (b) conditions. The relative values of somatostatin1 mRNA conditions. Means represented by different letters are significant expressions were normalized against β-actin and then averaged. (P < 0.05). Values are mean ± SEM Means represented by different letters are significant (P < 0.05). Values are mean ± SEM continuous light conditions (Fig. 3b). In some fishes, the rise of melatonin secretion during scotophase is con- during photophase (Peinado et al. 1990; Ishikawa et al. trolled using an endogenous clock. In pike Esox and zeb- 1997). We investigated biological rhythms of growth- rafish Danio rerio, the expression of the melatonin related genes in the diencephalon of the tiger puffer. synthesis gene (arylalkylamine N-acetyltransferase The expression patterns of ss1 mRNA were similar to (AANAT)) shows nocturnal rhythms in 12L:12D condi- those shown in previous studies. The ss1 mRNA expres- tions, and this gene has similar expression rhythms dur- sion in the diencephalon of tiger puffers increased dur- ing continuous light or dark conditions (Bégay et al. ing the dark phase in 12L:12D conditions (Fig. 4). 1998). These fish have circadian rhythms of high secre- However, ss1 mRNA expression rhythm disappeared tion during scotophase caused by the circadian oscilla- under 24L conditions. Therefore, ss1mRNA suggest to tion of the AANAT gene via an endogenous clock. have the daily rhythm according to light and dark. Therefore, we suggest that melatonin have the circadian In our presents, ghrh1 and ghrh2 mRNA exhibited diur- rhythm in the tiger puffer. nal rhythms in tiger puffers (Figs. 5 and 6). In rats, ghrh SS and GHRH regulate the synthesis and secretion of mRNA expression also exhibited diurnal rhythms (Bredow GH in the pituitary of vertebrates, including fish et al. 1996). Peak expression of hypothalamic ghrh mRNA (Bertherat et al. 1995; Sheridan and Hagemeister 2010). occurred during the early part of photophase in rats. The However, SS and GHRH rhythms in fish are not well ghrh expression gradually decreased up to scotophase, and understood since the majority of research has focused the mRNA expressions maintained low levels during sco- on mammals. Circadian variation of SS levels in rat tophase. Diurnal rhythms of hypothalamic ghrh mRNA plasma showed more elevation during scotophase than were also observed in rats (Gardi et al. 1999). In these Kim et al. Fisheries and Aquatic Sciences (2017) 20:17 Page 6 of 8 Fig. 5 Daily and circadian expressions of ghrh1 mRNA in the Fig. 6 Daily and circadian expressions of ghrh2 mRNA in the diencephalon of tiger puffer under 12L:12D (a) and continuous light diencephalon of tiger puffer under 12L:12D (a) and continuous light (b) conditions. The relative values of ghrh1 mRNA expressions were (b) conditions. The relative values of ghrh2 mRNA expressions were normalized against β-actin and then averaged. Means represented normalized against β-actin and then averaged. Means represented by different letters are significant (P < 0.05). Values are mean ± SEM by different letters are significant (P < 0.05). Values are mean ± SEM studies, hypothalamic ghrh levels increased rapidly in the expressions in tiger puffers. In our experiment, ss1 mRNA first hour after light onset, then decreased for 4 h after in diencephalon of tiger puffer was showed inducing expres- light onset. The ghrh level gradually increased up to sion by melatonini.p (Fig.7a).But,noeffectof melatonin the beginning of scotophase and then steadily de- on mRNA expressions of ghrh1 and ghrh2 was detected creased during scotophase. The ghrh1 and ghrh2 (Fig. 7b, c). This suggests that the daily fluctuation of ss1 mRNA rhythms in tiger puffers in this study were gene in the tiger puffer is controlled by melatonin, but not similar to the ghrh mRNA rhythms in rats. The ex- ghrh gene. Few studies have addressed the correlation of pression of ghrh1 mRNA in tiger puffers was high in melatonin with SS or GHRH in vertebrates, including mam- the early part of photophase and then decreased to- mals. Our review of the literature found studies only on the ward the midpoint of photophase. Further, the ghrh1 relationship of melatonin to SS in the rat hippocampus mRNA increased from the midpoint up to the end of (Izquierdo-Claros et al. 2004). This study showed a reduc- photophase. During scotophase, ghrh1 mRNA showed tion in SS levels through decreasing somatostatinergic sys- prolonged low expression levels. Therefore, we sug- tem activity, including inhibition of SS receptor activity by gest that tiger puffers have daily rhythms of ghrh ex- melatonin in the hippocampus. Although this finding seems pression that vary with light and dark conditions. to conflict with our results, we suggest that daily and circa- Our results showed that the ss1 mRNA expression pattern dian rhythms of growth-related genes are directly or indir- was similar to the pattern of melatonin secretion, but ghrh1 ectly controlled by melatonin in the tiger puffer. and ghrh2 mRNA expression patterns were opposite to Briefly, our results suggest that melatonin secretion and those of melatonin secretion. We then examined the correl- growth-related gene expressions follow daily and circadian ation of melatonin secretion and growth-related gene rhythms in the tiger puffer. As a result, melatonin showed Kim et al. Fisheries and Aquatic Sciences (2017) 20:17 Page 7 of 8 Acknowledgements This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2012R1A6A3A04041089). Funding This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF). Availability of data and materials Not applicable. Authors’ contributions BH and SP designed and carried out the data analysis and manuscript writing. SW participated in the fish sampling and data analysis. YT and AT participated in the plasma melatonin data analysis. YD participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. Ethics approval All experiments were conducted in compliance with both the Animal Care and Use Committee guidelines of the JeJu National University. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author details Marine Science Institute, Jeju National University, Jeju 6333, South Korea. Aquafeed Research Center, National Institute of Fisheries Science (NIFS), Pohang 37517, South Korea. Department of Electrical Engineering and Bioscience, School of Advanced Science and Engineering, Waseda University, Fig. 7 Expression of growth-related genes mRNA in the tiger puffer Shinjuku, Tokyo 162-8480, Japan. Department of Chemistry, Biology and diencephalon by after 1 h of melatonin i.p. Means represented by Marine Sciences, Faculty of Science, University of the Ryukyus, Nishihara, different letters are significant (P < 0.05). The relative values of (a) Okinawa 903-0213, Japan. Jeju International Marine Science Research & ss1, (b) ghrh1 and (c) ghrh2 mRNA expressions were normalized Logistics Center, Korea Institute of Ocean Science & Technology, Jeju 63349, South Korea. 19-5, Hamdeok 5(o)-gill, Jocheon, Jeju Special Self-Governing against β-actin and then averaged. 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