Program

Kisspeptin, encoded by the kiss1 gene, has the ability to activate its receptor, Kiss-R and known to play an essential role in vertebrate reproduction. We have previously identified two kisspeptin (kiss1 and kiss2) and two kisspeptin receptor types (Kiss-R1 and Kiss-R2) genes in the teleost species. In the zebrafish brain, kiss1 is expressed in the ventral habenula, while kiss2 is expressed in the preoptic-hypothalamic region. However, the functional significance of the existence of two kisspeptin types in the same animal species remains unclear. Recently, we have identified the potential role of Kiss1 in serotonergic modulation as well as in alarm substance (AS)-induced fear response in the zebrafish. Furthermore, inactivation of habenular kisspeptin neural pathway diminished AS-induced fear response, suggesting the neuromodulatory role of habenular kisspeptin neurons in fear pathway. On the other hand, the Kiss2 neural population in the hypothalamic nuclei has been viewed as a potent regulator of reproductive function in fish. We have also shown expression of Kiss-R2 in hypophysiotropic gonadotropin-releasing hormone (GnRH) neurons. Kiss2 neurons are sensitive to steroid hormones and to environmental temperature, factors that are important for the control of reproduction. These results suggest that the two kisspeptin systems may play independent roles in non-reproductive and reproductive functions.

Fish are exposed to various environmental stressors, such as pollutants and acute changes in temperature, and the chances of succumbing to infectious diseases may be increased as a result. Exposure of organisms to stressors may result in a series of biochemical and physiological changes in the body. At the organismal level, these changes are mediated by the neuroendocrine system. There is also a cellular stress response which is a characteristic feature of the induction of a family of special proteins following exposure to stressful situations. Although maintaining homeostasis should be a key process for coping with stress, changes in physiological set points would be an important aspect of the adaptive response, especially as a mechanism for coping with stressors. In the course of studies on the fish health in response to a stress, we found that stress caused by handling or chemicals regulates the expression of growth-related genes, such as growth hormone receptor (ghr) and insulin-like growth factor-1 (igf1) genes, in fish. In addition to these stress trials, we examined the redox state, such as levels of glutathione and lipid hydroperoxide, in response to heat shock or high doses of a dietary antibiotic (oxytetracycline, OTC). The results concerning the changing patterns of stress-related biomarkers indicate that both heat shock and dietary OTC induce oxidative stress, which might enhance oxidation in the body. Hence, antioxidative dietary supplements could suppress environmental stimuli-induced oxidative stress and improve the redox state in aquacultured fish. Furthermore, manipulation of positive stress (good stress or desirable stimuli, such as mild physiological or thermal treatment, osmotic stress, chemical stress, rearing density and light condition), might be useful to control fish fitness in terms of health, growth, the immune system, etc. The possibilities of using positive stress and avoiding negative stress (bad stress) for fish will also be discussed.

Insulin-like growth factor (IGF)-I is a multifunctional hormone involved in somatic growth, metabolism, osmoregulation, embryonic development and gonadal development in fish. IGF-I exerts its actions through endocrine, paracrine and autocrine pathways. Regardless of its mode of action, the activity of IGF-I is regulated by multiple IGF-binding proteins (IGFBPs). Six IGFBPs are present in mammals and they modulate IGF-I actions differently, adding another layer of complexity in the regulation of IGF-I actions. Moreover, some IGFBPs have actions independent of IGF. Unraveling functions of IGFBPs is therefore key to understand the mechanism of growth regulation. However, studies on fish IGFBPs especially at protein level are hampered by the fact that fish in general have two copies of each IGFBP and their identities are not well established. In addition, salmon have 19 IGFBP paralogs due to their tetraploid origin, making functional and physiological analyses more difficult. We have been focusing on endocrine IGF-I and IGFBPs in salmon because of their importance in integrating whole animal growth. This presentation summarizes what is currently known about circulating fish IGFBPs and discusses future direction. Three IGFBPs are consistently detected in salmon serum/plasma. We identified them through protein purification and cDNA cloning as IGFBP-1a, -1b and -2b, respectively. Both IGFBP-1a and -1b are induced under catabolic conditions such as stress and fasting and may inhibit IGF-I action. IGFBP-2b is a major carrier of circulating IGF-I like mammalian IGFBP-3 and its levels are high when fish are fed and growing. These findings suggest that salmon IGFBP-2b acquired the role and regulation similar to mammalian IGFBP-3. We are currently making functional recombinant salmon IGFBPs to examine their roles in growth.

We found that the Koi carp (Cyprinus carpio) undergoes morphological color changes, but scarcely physiological color changes, during the adaptation to background color. The number of melanophores in the scale decreases when the fish are acclimated to a white background color, whereby melanosomes are unable to completely aggregate in the melanophores. Melanosome translocation might be inhibited by defective melanophores or due to the lack of stimulation by hormonal or neuronal regulators in Koi carp. Melanin-concentrating hormone (MCH) is a neuropeptide, which generally causes melanosome aggregation in teleosts. We examined the function of MCH in the Koi carp and common carp. Intraperitoneal administration of MCH induced melanosome aggregation in both Koi and common carp, indicating that those melanophores are able to translocate melanosomes. Conversely, in vitro treatment of the scale with MCH, stimulated melanosome aggregation in common carp but not in Koi carp, suggesting that melanophores of Koi carp are not MCH sensitive. Melanocyte-stimulating hormone (MSH) induces melanosome dispersion in some vertebrates, including teleosts, through melanocortin receptors (MCRs). The results of reverse-transcription PCR indicated that mc1r and mc5r might be expressed in the scale of both Koi carp and common carp, suggesting that MSH might stimulate melanosome dispersion. Taken together, these results indicate that MCH of Koi carp might be involved in skin color changes by regulating endocrine factors, such as MSH, although MCH does not act directly on melanophores.

On full-life cycle aquaculture of some fish like bluefin tuna (Thunnus orientalis), cannibalistic behavior in their juvenile stage is a serious problem. Therefore, less aggressive breed race is beneficial for farming such species. Recently, genome editing which can knock out (KO) targeting gene or insert reporter gene into genome is expected as new breeding technology on aquaculture. This study was aimed to optimize the mutagenesis technique on chub mackerel (Scomber japonicus), same family of bluefin tuna, by using Transcription Activator-Like Effector Nucleases (TALEN). Chub mackerel has ideal characteristics for genome editing in scombroid fish, such as easy handling, long spawning season and maturation in one year. In this study, we tried to KO arginine-vasotocin receptor (AVTR-V1a2) where some reports suggest its involvement with aggressiveness on teleost. Partial avtr-v1a2 (740 bp) was obtained from genome and TALEN was designed to target one cysteine. TALEN was injected into fertilized eggs at different concentrations and these mutations were analyzed by heteroduplex mobility assay (HMA). In HMA analysis, all individuals showed mutation on 100 ng/ul concentration and random sequence analysis on PCR products from their genome showed 43.9% mutated cell rate. Additionally, we succeeded in collecting fertilized eggs in winter (out of spawning season) by controlling photoperiod and water temperature. Thus, we can conduct genome editing experiment on chub mackerel for half year at most. By the use of above methods, we can KO AVTR-V1a2 efficiently. The AVTR-V1a2 null character evaluation by behavior analysis software is under development. This work was supported by Council for Science, Technology and Innovation (CSTI), Cross-ministerial Strategic Innovation Promotion Program (SIP), “Technologies for creating next-generation agriculture, forestry and fisheries” (funding agency: Bio-oriented Technology Research Advancement Institution, NARO).

In our previous work, it was revealed that the final oocyte maturation (FOM) is induced by male-released sex pheromone in the honeycomb grouper. It was also found that FOM is indicated by injection of human chorionic gonadotropin (HCG). Thus, in the present study, we researched the changes in gene expression of GtHs (fshβ and lhβ) in the pituitary and their receptors (fshr and lhr) in the ovary during the process of FOM, in order to understand the physiological mechanisms from acquisition of maturational competence to completion of FOM.
Experimental fish were collected around Sesoko island, Okinawa, Japan. The females were stocked in an outdoor tank isolated from male fish until the start of the experiment. The females were divided into three experimental groups as follows: Male water (MW) group was reared with male rearing water, HCG group was injected with HCG, and control group was reared in seawater without males. Blood, ovary, and pituitary of the females were sampled after 0, 24, 48, and 72 hr from the start of the experiment. The gene expressions of GtHs and their receptors were detected by real-time PCR. In addition, plasma 17β-estradiol (E₂) and testosterone (T) levels were measured by ELISA.
The oocyte stage of all initial samples was the tertiary yolk stage and FOM was not yet initiated. In the control group, the tertiary yolk stage remained until end of the experiment. In HCG group, FOM was observed at 48 and 72 hr, and the plasma E₂ and T levels tended to increase, although there was no change in GtHs and their receptors gene expression levels. In the MW group, plasma E₂ and T levels, and lhβ gene levels increased, while FOM completed at 72 hr. On the other hand, in the gene expression levels of GtH receptors, only lhr increased at 24 hr in MW group.
These results suggest that the male pheromone induces FOM via secretion of LH. Interestingly, the pheromone also enhanced the gene expression of lhr prior to LH synthesis to induce the FOM in the honeycomb grouper.

Kisspeptin (Kiss1) is an endogenous ligand that modulates GnRH secretion through the interaction with a GPR54 (KissR1). In fish, the C-terminal pentadecapeptide amide, Kiss1-15, exerts the bioactivities. Our previous study showed that peripheral administration of Kiss1-15 strongly induced spermatogenesis in chub mackerel (cm: Scomber japonicus) and GnRH1 neurons co-expressed KissR1. Thus, Kiss1 peptide may be a potentially critical analog in the acceleration of gonadal development. In this study, first, we characterized the functional form of the Kiss1 peptide in chub mackerel; the deduced amino acid sequence indicates that core peptide is hexadecapeptide (Kiss1-16: HQDMSSYNFNSFGLRY-NH2). The result of SRE-Luc reporter assay showed that Kiss1-16 had stronger potency for receptor activation compared with Kiss1-15 or alanin (A) substitutional peptide (AQDM-16). This indicated that 16 length and histidine (H) are important for receptor activity. Next, we cloned the Kiss1 core sequences in other Scombroid fish (3 Scomber, 8 Thunnus, 1 Katsuwonus, 1 Scomberomorus, 1 Euthynnus, 1 Sarda and 2 Auxis). Nevertheless high homology (>86%), genus except Scomber fishes showed an absence of (H) at the N-terminus and indicate that the core peptide is 15 aa in length for these species. Furthermore, residues from 13 and 14 up-stream of C-terminus showed variation in interspecies. In the presented 17 species, five types of deduced core peptide forms were observed. Finally, we checked the compatibility of agonistic activity of these peptide types with or without the addition of (H) in interspecies of Scombroid family using SRE-Luc reporter system. For this study, we newly cloned kissr1 gene from Japanese spanish mackerel (jsm: Scomberomorus niphonius); it showed 95% homology with cmKissR1. All tested peptides showed high agonistic activity for both cmKissR1 and jsmKissR1, respectively. The present study may lead to a possible development of common Kiss1 analogs in artificial maturation of Scombroid fish.