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Using a recombinant chimeric single-chain follicle stimulating hormone (FSH), we established a radioimmunoassay (RIA) for red seabream (Pagrus major) FSH (pmFSH) which became a powerful tool for studying reproductive physiology. We studied the profiles in plasma and pituitary concentrations of FSH and luteinizing hormone (LH) during sexual maturation. A pre-established RIA for red seabream LH was used for the LH measurements. The regulation of FSH and LH secretion from the pituitary was investigated using a gonadotropin-releasing hormone analog (GnRHa) in vivo and in vitro. Marked differences in plasma and pituitary FSH levels were observed between males and females; pituitary FSH content in males was much higher than that in females during all seasons, and plasma FSH levels in males were high during the spawning season, whereas those in females were unchanged. In contrast, plasma and pituitary levels of LH were elevated before and during the spawning season in males and females. Injecting or implanting (cholesterol pellet) a GnRHa into adult and juvenile red seabream resulted in significant increases in plasma LH concentrations; however, no significant change was observed in plasma FSH. Moreover, GnRHa stimulated only LH secretion in an in vitro experiment using dispersed pituitary cells. The discrete FSH and LH secretion profiles revealed suggest differential roles for the two gonadotropins during red seabream gametogenesis. In addition, the marked difference in pituitary FSH levels in males and females suggests the relative significance of FSH in male reproduction.

In mammals, the role of the suprachiasmatic nucleus (SCN) as the primary circadian clock that coordinates the biological rhythms of peripheral oscillators is well known. However, in teleosts, it remains unclear whether the SCN also functions as a circadian pacemaker. We used in situ hybridization (ISH) techniques to demonstrate that the molecular clock gene, per2, is expressed in the SCN of flounder (Paralichthys olivaceus) larvae during the day and down-regulated at night, demonstrating that a circadian pacemaker exists in the SCN of this teleost. The finding that per2 expression in the SCN was also observed in the amberjack (Seriola dumerili), but not in medaka (Oryzias latipes), implies that interspecific variation exists in the extent to which the SCN controls the circadian rhythms of fish species, presumably reflecting their lifestyle. Rhythmic per2 expression was also detected in the pineal gland and pituitary, and aperiodic per2 expression was observed in the habenula, which is known to exhibit circadian rhythms in rodents. Since the ontogeny of per2 expression in the brain of early flounder larvae can be monitored by whole mount ISH, it is possible to investigate the effects of drugs and environmental conditions on the functional development of circadian clocks in the brain of fish larvae. In addition, flounder would be a good model for understanding the rhythmicity of marine fish. Our findings open a new frontier for investigating the role of the SCN in teleost circadian rhythms.

The gastric glands of marine aquaculture fish species are known to start secretion of pepsinogens (Pep) during larval development, far after first feeding. Although characterization of gastric gland development is considered useful for managing larval feeding, the timing of initial Pep synthesis by the gastric gland has only been reported for a few species. Consequently, this study employed in situ hybridization (ISH) to characterize the onset of expression of genes encoding the gastric enzymes Pep and Chitinase (Chi), as well as the complete development of gastric glands in the larval stomach of Japanese flounder ( Paralichthys olivaceus ), spotted halibut ( Verasper variegatus ), seven-band grouper ( Epinephelus septemfasciatus ) and greater amberjack ( Seriola dumerili ). The expression of both Chi and Pep was observed to begin in the cardiac region of the larval stomach at 25 days post-hatching (dph) in Japanese flounder, 36 dph in spotted halibut, 38 dph in seven-band grouper and 13 dph in greater amberjack. The gastric glands of these species were fully developed in the stomach approximately 5–12 days after the initiation of Pep expression. Development of gastric glands was observed to coincide with external manifestations of metamorphosis, such as the development of adult coloration in Japanese flounder and spotted halibut of the Pleuronectiformes, while it finished prior to adult-type coloration in seven-band grouper and greater amberjack of the Perciformes.

Studies on the cellulose utilization by animals have been conducted in keeping with the recent developments in molecular biology. In mollusks, endogenous cellulases have been reported from blue mussel, abalone, and freshwater snail. We previously reported the possibility of cellulose assimilation by Corbicula japonica , a representative bivalve dominant in brackish water environments in Japan, and the cloning of its endogenous cellulase (β-1,4-glucanase) gene (Sakamoto, K., Touhata, K., Yamashita, M., Kasai, A. and Toyohara, H., 2007. Cellulose digestion by common Japanese freshwater clam Corbicula japonica . Fish. Sci. 73, 675–683). However, the gene of β-glucosidase, another enzyme essential for the complete cellulose decomposition to glucose units, has not yet been isolated from the mollusk. Therefore, we attempted the molecular cloning of endogenous β-glucosidase from C. japonica and succeeded in the isolation of a cDNA with a 2832-bp open reading frame (ORF) encoding 943 amino acid residues ( CjCel1A ). CjCEL1A has 2 repeated GHF-1(Glycosyl Hydrolase family 1)-like domains and showed high similarity with known insect β-glucosidases and mammalian lactase-phlorizin-hydrolases. Reverse transcription (RT)-PCR analysis and in situ hybridization revealed that CjCEL1A is likely to be produced in the secretory cells in the digestive gland, suggesting that CjCEL1A is a digestive β-glucosidase of C. japonica and is not derived from symbionts.

Preparation of high quality eggs is essential in aquaculture. While egg quality is widely regarded as being largely dependent on the nutrients that are stored in the yolk, the identity of the factors responsible for affecting this egg quality is not yet known. Thus, in order to ensure the quality of better quality eggs, it is essential to elucidate how the nutrients in yolk influence embryogenesis. Using a microinjection system, we performed two tests with zebrafish embryos to evaluate the effects of nutrients on embryogenesis. To determine whether retinoic acid, excess retinoids and carotenoids in yolk cause malformations, we injected retinoic acid, retinal, retinol and β-carotene into the yolk of 1–4 cell cell-stage embryos, and compared their effects on embryogenesis. The injection of retinal into yolk causes similar anomalies to those induced by retinoic acid in the brain, jaw, heart, as well as suppressing hatching rate. Retinol had a lower tetragenic effect than retinal and toxicity was not detected for β-carotene. These findings suggest that excess retinal and retinol in yolk can impair egg quality by causing malformations and decreasing hatching rate. We then compared the protective effects of astaxanthin, β-carotene and α-tocopherol against oxidative stress in embryos. After injecting one of these compounds into the yolk of a 1–4 cell-stage embryo, embryos were exposed to H 2 O 2 for 2 h from 24 h post-fertilization (hpf) and mortality and apoptosis were observed at 48 hpf. Astaxanthin conferred embryos with a marked tolerance against mortality and apoptosis due to H 2 O 2 exposure, suggesting its potential application to the improvement of egg quality by increasing the tolerance of embryos toward oxidative stress. The methodology presented herein describes a quick method for evaluating the effects of nutrients on egg quality.

Leptin is a key factor for the regulation of food intake and energy homeostasis in mammals, but information regarding its role in teleosts is still limited. There are large differences between mammalian and teleost leptin at both gene and protein levels, and in order to characterize the function of leptin in fish, preparation of species-specific leptin is therefore a key step. In this study, full-length cDNA coding for rainbow trout leptin was identified. In spite of low amino acid sequence similarity with other animals, leptin is highly conserved between trout and salmon (98.7%). Based on the cDNA, we produced pure recombinant trout leptin (rt-leptin) in E. coli , with a final yield of 20 mg/L culture medium. We then examined the effects of intraperitoneal (IP) injection of rt-leptin on feeding behavior and gene expression of hypothalamic NPY and POMCs (POMC A1, A2 and B) in a short-term (8 h) experiment. The rt-leptin suppressed food intake and led to transient reduction of NPY mRNA levels, while the expression of POMCs A1 and A2, was elevated compared with vehicle-injected controls. These results for rainbow trout are the first that describe a physiological role of leptin using a species-specific orthologue in teleosts, and they suggest that leptin suppresses food intake mediated by hypothalamic regulation. This anorexic effect is similar to that observed in mammals and frogs and supports that the neuroendocrine pathways that control feeding by leptin are ancient and have been conserved through evolution.

We identified cDNA coding for a homologue to mammalian leptin in puffer, Takifugu rubripes, using genomic synteny around the human leptin gene. In addition to significant sequence homologies, the puffer leptin (pLEP) displays characteristic structural features in common with mammalian leptin. The pLEP mRNA was expressed mostly in the liver that contained abundant lipids. In addition, homologues to pLEP were found in the databanks for three fish species (salmon, medaka, and Tetraodon) and two amphibians (salamander and Xenopus). The phylogenetic analysis shows rapid rates of molecular divergence among leptins from different vertebrate classes, but not between mammals and avians.

We have cloned for the first time two cDNAs encoding distinct types of tissue inhibitor of metalloproteinases-2 (TIMP-2) from teleost fish, Japanese flounder, and designated these types as jfTIMP-2a and jfTIMP-2b. The open reading frames of the jfTIMP-2a and jfTIMP-2b cDNAs are composed of 663 and 657 nucleotides and 221 and 219 amino acids, respectively. Both jfTIMP-2s contain 12 cysteine residues, which might form six disulfide bonds as in other animals' TIMP-2s. The predicted full-length amino acid sequence of jfTIMP-2a has lower identity to jfTIMP-2b (63%) than to those of human (74%) and chicken (73%) TIMP-2s, but higher than to those of other human TIMPs (TIMP-1: 39%, TIMP-3: 43%, TIMP-4: 45%), indicating that jfTIMP-2a is a common TIMP-2, while jfTIMP-2b is unique to Japanese flounder. However, the C-terminal region including the last three disulfide bonds of jfTIMP-2b has higher amino acid identity to those of other animal TIMP-2s than to that of jfTIMP-2a. Reverse-transcribed polymerase chain reaction (RT-PCR) analysis showed the mRNAs of jfTIMP-2a and jfTIMP-2b to be ubiquitously expressed in all tissues examined, but with different expression patterns. These findings suggest that the two distinct jfTIMP-2s might perform different functions in teleost tissues.