Search

Genome editing is a technology that can remarkably accelerate crop and animal breeding via artificial induction of desired traits with high accuracy. This study aimed to develop a chub mackerel variety with reduced aggression using an experimental system that enables efficient egg collection and genome editing. Sexual maturation and control of spawning season and time were technologically facilitated by controlling the photoperiod and water temperature of the rearing tank. In addition, appropriate low-temperature treatment conditions for delaying cleavage, shape of the glass capillary, and injection site were examined in detail in order to develop an efficient and robust microinjection system for the study. An arginine vasotocin receptor V1a2 (V1a2) knockout (KO) strain of chub mackerel was developed in order to reduce the frequency of cannibalistic behavior at the fry stage. Video data analysis using bioimage informatics quantified the frequency of aggressive behavior, indicating a significant 46% reduction (P = 0.0229) in the frequency of cannibalistic behavior than in wild type. Furthermore, in the V1a2 KO strain, the frequency of collisions with the wall and oxygen consumption also decreased. Overall, the manageable and calm phenotype reported here can potentially contribute to the development of a stable and sustainable marine product.

Kisspeptin is an endogenous ligand that modulates gonadotropin-releasing hormone (GnRH) secretion and plays an essential role in the reproduction of mammals. In fish, two subtypes of kiss genes (kiss1 and kiss2), which encode kisspeptins, have been reported. Our previous study showed that kisspeptin 2 receptor (KissR2) is highly expressed in the vicinity of GnRH1 neurons, and that centrally administered Kiss2, but not Kiss1, significantly increased gonadotropin synthesis in chub mackerel Scomber japonicus. The results of this latter study suggested that the kisspeptin system plays an important role in chub mackerel reproduction, and that Kiss2 in particular plays a dominant role in the regulation of the central reproductive axis. In the present study, we identified the functional form of Kiss2 peptide in 16 species of Scombridae. All the species possessed kiss2, and the deduced mature peptide had 12 amino acids, all of which were very highly conserved, except for the second amino acid from the N-terminal end that was found to vary across species. However, species specificity of the second residue did not affect signal transduction in chub mackerel KissR2, bluefin tuna Thunnus orientalis KissR2, or Japanese Spanish mackerel Scomberomorus niphonius KissR2. Therefore, the importance of the Kiss2 system, shown in studies of chub mackerel reproduction, may be relevant across all species of Scombridae.

We analysed the data of swordtip squid Uroteuthis edulis caught in the eastern Tsushima Strait, from April to September between 2012 and 2018, to consider the relationships among mantle length and ...

For seasonal breeders, photoperiodic changes are important signals that mark the start of the breeding season. Thyroid-stimulating hormone (TSH) is a glycoprotein hormone that not only promotes the secretion of thyroid hormone but also plays a key role in regulating seasonal reproduction in birds and mammals. However, whether TSH activation has been implicated as a seasonal indicator in fish breeding has not been fully investigated. In this study, we isolated tshb as a starting point to elucidate the effect of photoperiodic changes on the activation of the reproductive axis of chub mackerel. The isolated tshb was classified as tshba, which is widely conserved in vertebrates. The quantitative PCR results showed that tshb was strongly expressed in the pituitary. When female and male chub mackerel with immature gonads were reared for six weeks under different photoperiodic conditions, the gonads developed substantially in the long-day (LD) reared fish compared to those in the short-day reared fish. Real-time PCR results showed that the expression level of tshb in the pituitary gland was significantly elevated in the LD group. Although there was no difference in the gonadotropin-releasing hormone 1 gene expression level in the preoptic area of the brain, follicle-stimulating hormone and luteinizing hormone gene expression levels in the pituitary were also significantly elevated in the LD group. In conclusion, TSH is a potential mediator of seasonal information in the reproductive endocrine axis and may induce gonadal development during the breeding season of chub mackerel.

The pituitary gonadotropins (Gths), follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), play critical roles in regulating gonadal development and sexual maturation in vertebrates. We developed non-competitive enzyme-linked immunosorbent assays (ELISAs) to measure Fsh and Lh in chub mackerel Scomber japonicus, which is a commercially important scombrid species. Mouse monoclonal antibodies specific for Fsh and Lh, and a rabbit polyclonal antibody against both Gths were produced by immunization with hormones purified from chub mackerel pituitaries. These monoclonal and polyclonal antibodies were used as capture and detection antibodies in the developed sandwich ELISAs. The ELISAs were reproducible, sensitive, and specific for chub mackerel Fsh and Lh. Parallelism between the standard curve and serial dilutions of chub mackerel serum and pituitary extract was observed for both Fsh and Lh ELISAs. Comparison between vitellogenic and immature females revealed that Fsh is secreted during vitellogenesis and Lh is barely released during immaturity. After gonadotropin-releasing hormone analog (GnRHa) injection, vitellogenic females showed increases in serum Lh, whereas serum levels of Fsh did not vary. Moreover, the serum steroid profiles revealed that estradiol-17β was continuously produced after GnRHa treatment, whereas 17,20β-dihydroxy-4-pregnen-3-one secretion was transiently induced. These results indicate that, in vitellogenic females, GnRHa stimulates the release of Lh, but not Fsh, which results in acceleration of vitellogenesis and induction of oocyte maturation via steroid production.

A comprehensive experimental system for Japanese anchovy, a promising candidate model organism for marine teleosts, was established. Through the design of a rearing/spawning facility that controls the photoperiod and water temperature, one-cell eggs were continuously obtained shortly after spawning throughout the rearing period. The stages of eggs are indispensable for microinjection experiments, and we developed an efficient and robust microinjection system for the Japanese anchovy. Embryos injected with GFP mRNA showed strong whole-body GFP fluorescence and the survival rates of injected- and non-injected embryos were not significantly different, 87.5% (28 in 32 embryos) and 90.0% (45 in 50 embryos), respectively. We verified that the Tol2 transposon system, which mediates gene transfer in vertebrates, worked efficiently in the Japanese anchovy using the transient transgenesis protocol, with GFP or DsRed as the reporter gene. Finally, we confirmed that genome-editing technologies, namely Transcription Activator-Like Effector Nucleases (TALEN) and Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR)/Cas9, were applicable to the Japanese anchovy. In practice, specific gene-disrupted fishes were generated in the F1 generation. These results demonstrated the establishment of a basic, yet comprehensive, experimental system, which could be employed to undertake experiments using the Japanese anchovy as a model organism for marine teleost fish.

Swordtip squid (Uroteuthis edulis) of various sizes are caught on an almost daily basis from early spring to late autumn in the eastern Tsushima Strait and southern Sea of Japan. In this study, we estimated the migratory routes of different sized squid jigged in the Tsushima Strait using statolith analyses, information on seawater temperature distributions and currents, and tracer experiments. Estimates of age and historic water temperatures suggested that both small and large squid hatch in the southern East China Sea and are then transported northeastward with the currents to the northern Kuroshio region. Following this, the small squid are transported north-northwestward to offshore waters, from where many of the squid enter the southern Sea of Japan and some move into coastal waters in the eastern strait, whereas large squid slowly move northward from the northernmost part of the Kuroshio region. Based on these distributions, it is likely that the small squid remain immature due to insufficient water temperatures over shorter durations, whereas the large squid mature in the Kuroshio region. Since migratory routes affect fishing ground formation, this information is important for both efficient fishing operations and stockmanagement of this species.

Gonadotropin-inhibitory hormone (GnIH), a neuropeptide, suppresses gonadotropin (GTH) secretion in birds and mammals. In fish, the GnIH homolog LPXRFamide (LPXRFa) produces mature peptides with species-dependent effects on sexual reproduction. Here, we investigated the effects of LPXRFa on GTH secretion in the chub mackerel (cm; Scomber japonicus). We cloned cmlpxrfa (603 bp) and cmlpxrfa-r (1416 bp). Additionally, we isolated lpxrfa from the bluefin tuna (Thunnus orientalis) to confirm the conservation of the LPXRFa mature sequence. Phylogenetic analysis showed that the LPXRFa precursor protein produces three mature peptides, LPXRFa-1, -2, and -3, in both species. Reverse transcription-quantitative polymerase chain reaction revealed that cmlpxrfa is expressed in the hypothalamus and thalamus and midbrain (T.MB), and sexual differences were observed. Receptor expression was observed in the preoptic area, hypothalamus, T.MB, and pituitary. Female hypothalamic lpxrfa expression did not change during puberty. Reporter gene assay showed that LPXRFa induced receptor activation via the CRE and SRE signaling pathways. However, in the presence of forskolin, an intracellular cyclic AMP enhancer, none of the LPXRFa could suppress receptor activity. The in vitro bioassay results showed that gonadotropin-releasing hormone-1 (GnRH1) had no effect on follicle-stimulating hormone (FSH) secretion, whereas the three LPXRFa significantly increased FSH secretion in pituitary cells from male chub mackerel. Contrarily, GnRH1 and three LPXRFa significantly increased luteinizing hormone secretion. The in vivo administration of LPXRFa had no effect on fshb and lhb expression in prepubertal and mature male chub mackerel. Overall, cmLPXRFa lacks the ability to suppress GTH secretion but can promote GTH secretion. Summary sentence The chub mackerel LPXRFamide gene produces three mature peptides and not have the ability to suppress gonadotropin secretion but may promote gonadotropin secretion.

In contrast to the swordtip squid (Uroteuthis edulis) caught in the spring and summer, those caught in the autumn in the Japan Sea and the Tsushima Strait are characterized by thick bodies with lar...

Chub mackerel (Scomber japonicus Houttuyn) is one of the most commercially important scombroid fish used as a food resource. Recently, there has been a demand for efficient rearing methods of this fish for a full-life cycle aquaculture. In the present study, we evaluated the physiological responses in the juvenile S. japonicus to different ambient salinities. A significantly higher gain of the body mass was observed in the juveniles reared in 24 g/L and 13 g/L seawater than in those reared in natural seawater (34 g/L) within 40 days of the experimental period without affecting mortality. A principal enzyme for osmoregulation, Na+/K+-ATPase, was expressed in the ionocytes located in the gill filaments of the juveniles. The number and the cell size of ionocytes and the enzymatic activity of Na+/K+-ATPase in the gills decreased within 10 days after the low-salinity challenge, which implies the reduction of the energy-consuming active ion secretion under the low-salinity environment. The physiological capacity for adaptation to low-salinity seawater in chub mackerel could be basic knowledge to carry out culturing of these fish in coastal sea pens where ambient salinity fluctuates. The improvement of the growth performance by rearing in low-salinity seawater will contribute to the efficient production of the seed juveniles for aquaculture.

The gonadotropin-releasing hormone (GnRH) is essential during pubertal onset, for its regulation of the synthesis and release of pituitary gonadotropins. Its action is mediated by GnRH receptors (GnRHRs) in the pituitary gonadotrophs. Our previous study demonstrated that the chub mackerel brain expresses three GnRH forms (gnrh1, gnrh2, and gnrh3), and that only GnRH1 neurons innervate anterior pituitary regions. Furthermore, chub mackerel gnrh1 mRNA exhibited a significant increase at pubertal onset. The present study aimed to isolate the functional GnRHR form involved in chub mackerel puberty. The open reading frame of our cloned receptor encodes 428 amino acids and contains seven transmembrane domains. Phylogenetic analysis also indicated clustering with other teleost-type IIB GnRHRs, mainly those involved in reproduction. Reporter gene assay results showed that all four synthetic peptides (GnRH1, GnRH2, GnRH3, and GnRH analogue) bind to the cloned receptor. Three deduced GnRH ligands stimulated luteinizing hormone (LH) release from cultured pituitary cells in vitro. Receptor gene expression was mainly detected in the pituitary and showed an increasing trend in the developing gonadal stages of both sexes during the pubertal process; this process was synchronous with previous studies of follicle-stimulating hormone beta (fshβ) and lhβ gene expression in chub mackerel. These results suggest that the cloned receptor is likely involved in the regulation of pubertal onset in this species. Therefore, we have designated the receptor cmGnRHR1.

Vitellogenenesis is a physiological process common in oviparous animals. The molecular profile, modifications, and utilization of vitellogenin (VTG), a precursor of yolk protein, have been characterized in various taxa to understand oogenesis within different modes of reproduction. Hormonal regulation of VTGs has been investigated in invertebrates, such as insects and crustaceans; conversely, little is known for cephalopods. In this study, we isolated two VTG genes (ue-VTG1 and ue-VTG2) from the loliginid swordtip squid, Uroteuthis edulis, via a comprehensive survey of a transcriptome database and subsequent cDNA cloning. Structural analysis of the two ue-VTGs revealed their unique features, namely the absence of two domains usually found in VTGs from other organisms: the von Willebrand factor D domain (vWD) and the domain of unknown function 1943 (DUF1943). Levels of ue-VTG1 and ue-VTG2 transcripts in the ovary, specifically in follicular cells, increased during the late-vitellogenic phase, suggesting that yolk accumulation progresses via paracrine interactions involving follicular cells and oocytes. N-terminal amino acid sequencing of biochemically purified yolk protein revealed its origins from these two VTGs, indicating that both are functional precursors of yolk protein. These results provide information that is essential to understanding the physiological pathway of yolk synthesis, accumulation, and storage in loliginid squids.

Here we report that the Kiss1 hexadecapeptide (Kiss1-16) directly regulates the functional form of gonadotropin-releasing hormone (GnRH) in the preoptic area (POA) of a scombroid fish model. In this study, we analyzed the localization of two kisspeptin (kiss1 and kiss2) neurons and two kisspeptin receptors (kissr1 and kissr2) in the brain of adult chub mackerel using in situ hybridization to determine whether the kisspeptin receptors co-localize with GnRH1 neurons. The kiss1- and kiss2-expressing neurons were mainly localized in the nucleus recessus lateralis (NRL) and the nucleus of the posterior recess (NRP) in the hypothalamus. Kissr1 was present in the anterior POA and the habenular nucleus. Kissr2 was widely distributed, including in the POA, lateral tuberal nucleus, NRL, and NRP. Notably, GnRH1 was expressed in neurons in the POA, and these neurons co-expressed kissr1. In contrast, kissr2 was expressed abundantly in the vicinity of GnRH1 neurons, but their co-expression did not seem to occur. We also characterized the endogenous mature form of the Kiss1 peptide. An in vitro reporter gene assay clearly showed that Kiss1-16 (HQDMSSYNFNSFGLRY-NH2) was more potent at receptor activation than Kiss1 pentadecapeptide (Kiss1-15), which is the form of Kiss1 found in other fish species. This study strongly suggests that kisspeptin signaling, especially Kiss1 signaling, is important for regulating reproduction in scombroid fish.

Leptin directly regulates kisspeptin neurons in the hypothalamus and gonadotropin secretion from the pituitary, making it a central player in the onset of mammalian puberty. Recently, we identified two leptin genes (lepa and lepb) and a single leptin receptor (lepr) in the marine perciform fish chub mackerel; however, the expression of these genes did not correlate with the expression of important reproductive genes or ovarian stage during female puberty. Here, we expand upon these initial observations by evaluating the expression of lepa, lepb, and lepr during pubertal transition and under differential feeding conditions in the male chub mackerel. Reverse transcription-polymerase chain reaction (RT-PCR) showed that lepa was primarily expressed in the liver of pubertal and gonadal recrudescence adults, as well as in the brain of adult fish; lepb was primarily expressed in the brain of all fish tested; and lepr was widely expressed in a variety of tissues. qRT-PCR analyses revealed significant increases in the hepatic expression of lepa in accordance with testicular stage, whereas pituitary follicle-stimulating hormone (fshβ) expression increased in unison with hepatic lepa. In contrast, expression of both brain lepa and lepb dramatically decreased during pubertal transition, with brain kisspeptin 1 (kiss1) expression strongly correlating with leptin expression patterns. In pre-pubertal males, lepa, lepb, and lper gene expression in the brain, pituitary gland, and liver decreased in fish given a high feed diet, relative to the controlled feeding group. Taken together, these results indicate high sexual specificity of leptin expression, suggesting a possible role for leptin signaling in endocrine and neuroendocrine functions during spermatogenesis in the pubertal male chub mackerel.

Leptin transmits information about energy stored in the periphery to the reproductive axis and is an essential signal for puberty initiation in mammals; however, to date, few studies have focused on the direct effects of leptin stimulation on reproductive factors in fish. This study demonstrated the effect of leptin stimulation on important reproductive factors and ovarian development in the marine teleost chub mackerel (Scomber japonicus). We prepared recombinant leptin and conducted functional analyses through in vitro bioassays using primary pituitary cells, long-term leptin treatment administered to pre-pubertal females, and intracerebroventricular (ICV) administration. The results showed that leptin stimulation strongly induced gonadotropin (follicle-stimulating hormone: FSH and luteinizing hormone: LH) secretion from pituitary cells collected from pre-pubertal females, and that long-term leptin treatment significantly promoted ovarian development and triggered pubertal onset. Furthermore, ICV administration of leptin did not affect kisspeptin gene expression but significantly upregulated gonadotropin-releasing hormone 1 (gnrh1), fshb and lhb gene expression in sexually immature females. These results strongly suggest leptin as an important signal for reproductive-axis activation in chub mackerel.

Leptin directly influences gonadotropin (GTH) secretion from female pituitary cells in vitro and is a key signal at the onset of puberty in female chub mackerel (Scomber japonicus). Here, we investigated whether leptin also influences GTH secretion in male chub mackerel. The addition of 1 nM homologous recombinant leptin to pre-pubertal male pituitary cells stimulated follicle-stimulating hormone secretion after 1 and 2 h of culture. Therefore, leptin signaling could also directly facilitate GTH secretion in male chub mackerel.

We investigated the monthly numbers of tracers moving from the southern East China Sea to the east side of Japan via the Sea of Amakusa, on the west side of Japan, by tracer experiments. We estimated the migratory route of large Swordtip squid (Uroteuthis edulis) caught in Sagami Bay, on the east side of Japan, by the micro-increment counts and variation of strontium:calcium ratios of the statoliths to test our hypothesis that the Sea of Amakusa plays the role of a distributor of this species to the east and west sides of Japan. Results of the monthly numbers of the tracers were generally consistent with the season of peak catches on the east side. The older tracers reaching the east side of Japan were found to have passed through the Sea of Amakusa at larger rates. Moreover, the estimated empirical water temperature history of the large squid caught in Sagami Bay showed two peaks, in winter and early summer, implying that the squid experienced the Kuroshio current twice and that during spring, they stayed in waters whose temperature was a few degrees lower than that of the Kuroshio; that is, in the Sea of Amakusa. Frequent and extensive marine observations will be needed to fully understand the distribution of this species around Japan.

Chronic Kiss1 administration strongly promotes gonadal development in immature chub mackerel (cm) (Scomber japonicus). Here, we performed an Alanine scanning (Ala-scanning) of Kiss1 to determine its key residues. Additionally, we examined functional peptides from 16 Scombridae species to develop maturation-inducing super-analogs that can be used universally in Scombridae species. In the Ala-scanning of Kiss1-15 (QDMSSYNFNSFGLRY), substitution of Gln1 and Asp2 did not affect agonistic activity. This suggests that peptides could be downsized. Furthermore, it is possible that Phe8 can be substituted by unnatural amino acids that are difficult to degrade. In molecular cloning, only Scomber showed a 16-residue form as a putative mature peptide. The other genera, did not have a His residue at the N-terminal, which indicated that the functional peptide was 15 residues and the second and third residues from the N-terminal showed variation between interspecies. Next, we examined the binding affinity of various synthetic Kiss1 core peptides in Scombridae interspecies using an SRE-Luc reporter system. We cloned Kiss1 receptors (KissR1) from bluefin tuna (bft) (Thunnus orientalis) and Japanese Spanish mackerel (jsm) (Scomberomorus niphonius) for the first time. In binding affinity with cm KissR1, bft KissR1, and jsmKissR1, the species specificity of the second residue from the N-terminus in each ligand could be ignored, but the difference in the third residue strongly affected receptor binding. Scombridae species possess the same Kiss1 system but the structure of the functional peptide might be species-specific.

Kisspeptin, a novel neuropeptide product of the Kiss1 gene, activates the G protein-coupled membrane receptor G protein-coupled receptor 54 (now termed Kiss1r). Over the last 15 years, the importance of the kisspeptin system has been the subject of much debate in the mammalian research field. At the heart of the debate is whether kisspeptin is an absolute upstream regulator of gonadotropin-releasing hormone secretion, as it has been proposed to be the master molecule in reproductive events and plays a special role not only during puberty but also in adulthood. The teleostean kisspeptin system was first documented in 2004. Although there have been a number of kisspeptin studies in various fish species, the role of kisspeptin in reproduction remains a subject of controversy and has not been widely recognized. There is an extensive literature on the physiological and endocrinological bases of gametogenesis in fish, largely derived from studying small, model fish species, and reports on non-model species are limited. The reason for this discrepancy is the technical difficulty inherent in developing rigorous experimental systems in many farmed fish species. We have already established methods for the full life-cycle breeding of a commercially important marine fish, the chub mackerel (cm), and are interested in understanding the reproductive function of kisspeptins from various perspectives. Based on a series of experiments clarifying the role of the brain-pituitary-gonad axis in modulating reproduction in cm, we theorize that the kisspeptin system plays an important role in the reproduction of this scombroid species. In this review article, we provide an overview of kisspeptin studies in cm, which substantially aids in elucidating the role of kisspeptins in fish reproduction.

In recent years, brain kisspeptin system has been shown to be involved in diverse reproductive function, including sexual differentiation in vertebrates. Our previous reports demonstrated that the chub mackerel (Scomber japonicus) brain expresses two kisspeptin (kiss1, kiss2), two kisspeptin receptor (kissr1, kissr2) and three gonadotropin-releasing hormone (gnrh1, gnrh2, gnrh3) genes. In the present study, using quantitative real-time PCR (qRT-PCR) assays, we analysed expression changes of these genes during early development (0-30dphs) and gonadal sex differentiation periods (37-60dphs). Absolute expression level of kiss-kissr-gnrh in the whole head was higher between 0 and 15dphs, in comparison to later developmental periods. Histological analyses revealed presence of sexually differentiated males and females with testicular and ovarian features at 37, 45, and 60dphs. In both males and females, kiss2, kissr1, and kissr2 levels were higher at 37dph, in comparison to 45 and 60dphs, with kiss1 showing no significant differences. Levels of all three gnrh mRNAs were higher at 45dph, in comparison to 60dph. Changes in the expression level of kiss-kissr-gnrh mRNAs in different brain regions of sexually differentiated males and females indicated differences in their regional distribution. These results suggest possible involvement of Kiss-KissR-GnRH systems during early development and gonadal sex differentiation in the chub mackerel.

Despite the commercial importance of swordtip squid Uroteuthis edulis in Tsushima Strait, which lies between southwestern Japan and South Korea, data on its hatching grounds and migratory routes are lacking. To infer this information, we first revealed a significant negative correlation between ambient water temperatures and strontium:calcium (Sr:Ca) ratios in squid statoliths through tank experiments. Next, we counted statolith microincrements in spring and summer migratory groups of the species to elucidate their hatching dates, after measuring Sr:Ca ratios from the nucleus to the edge of each statolith. Based on these methods, spring and summer groups appear to inhabit the southern East China Sea as juveniles, and move northeastward with currents along shelf edges in the Kuroshio region. However, individuals in the spring group are apparently affected by cold waters in the northern East China Sea before reaching the Tsushima Strait, likely resulting in the unique characteristics of this seasonal group. In contrast, those in the summer group are likely unaffected because of higher water temperatures in summer than in spring. This study is the first to infer the migratory routes of the spring and summer groups of the swordtip squid.

Kisspeptin (Kiss) and its cognate receptor (Kiss1R), implicated in the neuroendocrine control of GnRH secretion in mammals, have been proposed to be the key factors in regulating puberty. However, the mechanisms underlying the initiation of puberty in fish are poorly understood. The chub mackerel Scomber japonicus expresses two forms of Kiss (kiss1 and kiss2) and two Kiss receptor (kissr1 and kissr2) genes in the brain, which exhibit sexually dimorphic changes during the seasonal reproductive cycle. This indicates that the kisspeptin system plays an important role in gonadal recrudescence of chub mackerel; however, the involvement of the kisspeptin system in the pubertal process has not been identified. In the present study, we examined the mRNA expression of kiss1, kiss2, kissr1, kissr2, and gnrh1 (hypophysiotropic form) in the brain of a chub mackerel during puberty. In male fish, kiss2, kissr1 and kissr2 levels increased significantly at 14weeks post-hatch (wph), synchronously with an increase in type A spermatogonial populations in the testis; kiss2 and gnrh1 levels significantly increased at 22wph, just before the onset of meiosis in the testes. In female fish, kiss2 increased significantly at 14wph, synchronously with an increase in the number of perinucleolar oocytes in the ovary; kiss1 and kiss2 levels significantly increased concomitantly with an increase in the kissr1, kissr2, and gnrh1 levels at 24wph, just before the onset of vitellogenesis in oocytes. The present results suggest positive involvement of the kisspeptin-GnRH system in the pubertal process in the captive reared chub mackerel.

To better understand the physiological mechanisms underlying reproductive dysfunction, such as impaired vitellogenesis and final oocyte maturation, we assessed endocrinological differences between captive and wild female jack mackerel (Trachurus japonicus). The presence of three gonadotropin-releasing hormone (GnRH) peptides was immunologically evaluated in jack mackerel brain tissues. Full-length cDNAs encoding GnRHs (gnrh1, gnrh2, and gnrh3) and gonadotropin subunits (fshb, lhb, and gpa) were cloned, sequenced, and quantitatively assayed. Of the captive females, 60% failed to undergo vitellogenesis, displaying immature (IM) or atretic oocytes, whereas 80% of wild females were captured during late vitellogenesis (LV) or ovulation (OV). The gnrh1 expression was significantly lower in captive fish than in wild LV and OV fish, while there were no significant differences in the expression of gnrh2 or gnrh3. The expression of fshb was lower in captive IM fish than in wild LV fish, but no significant differences were observed between the captive IM and LV individuals. The lhb expression was elevated in the wild LV and OV fish, and gpa expression was greatest in the wild OV fish. Serum estradiol-17β levels were significantly lower in captive IM fish than in captive LV fish. The results indicate that captive-rearing stress may impair vitellogenesis and negatively influence the transcription of gnrh1 in the brain and GtH synthesis in the pituitary.

The kisspeptin receptor (Kiss1R) is a cognate receptor for kisspeptin (Kiss), and this Kiss-Kiss1R system has been shown to regulate seasonal reproduction in vertebrates. Our previous study found the chub mackerel (Scomber japonicus) brain expresses both kiss1 and kiss2 and exhibits sexually dimorphic changes during the seasonal reproductive cycle. The present study cloned two subtypes of kissr from the chub mackerel brain, and their signal transduction pathways to Kiss1 and Kiss2 were characterized in a mammalian cell line. Results of identification showed that kissr1 and kissr2 mRNAs encode 369 and 378 deduced amino acids, respectively, and share 52% similarity in amino acid sequences. In vitro functional analysis revealed that chub mackerel Kiss receptor signals are also preferentially transduced via the protein kinase C (PKC) rather than protein kinase A (PKA) pathway. Synthetic chub mackerel Kiss1-15 and Kiss2-12 peptides showed the highest potency for the activation of KissR1 and KissR2, respectively, stronger than their corresponding Kiss-10 peptides. Tissue distribution analyses indicated that both genes are highly expressed in the brain and that only kissr2 mRNA is expressed in the pituitary of both sexes. Unexpectedly, both kissr1 and kissr2 mRNAs were detected only in the testes. Seasonal expression changes showed higher expression levels of both kissr1 and kissr2 mRNAs in the brain of females during the early vitellogenic period; however, no significant differences were found in the brain of males. Pituitary kissr2 mRNA levels showed no significant variations. In the testes, the kissr1 mRNA expression level increased dramatically at spermiation compared with the immature and post-spawning periods. However, kissr2 mRNA levels in the testes did not vary significantly at different testicular stages. These results suggest that both kissr1 and kissr2 likely participate in the seasonal ovarian development of females, and thus in males, we propose a paracrine or autocrine role for kissr1 in testicular development.

The gonadosomatic index (GSI) is widely used as a simple measure of reproductive capacity, but its validity has often been questioned. This study showed the inter-spawning variation in the predicted GSI of Japanese anchovy using the gravimetric oocyte packing density method. Time course sampling showed that final oocyte maturation and subsequent ovulation occurred from afternoon to evening on the day of spawning. Oocyte in vitro assay, however, suggested that the timing of the spawning could be predetermined by midnight the day before spawning. Models predicted that the GSI of a female just after spawning gradually increases until the completion of germinal vesicle breakdown, after which it dramatically rises by 24–53 % per hour within a few hours of the onset of ovulation. This indicates that the predicted GSI of a female increases by 3.5 times within about 19 h before ovulation, even if the relative batch fecundity remains constant.

The nuclear envelopes surrounding the oocyte germinal vesicles of lower vertebrates (fish and frog) are supported by the lamina, which consists of the protein lamin B3 encoded by a gene found also in birds but lost in the lineage leading to mammals. Like other members of the lamin family, goldfish lamin B3 (gfLB3) contains two putative consensus phosphoacceptor p34cdc2 sites (Ser-28 and Ser-398) for the M-phase kinase to regulate lamin polymerization on the N- and C-terminal regions flanking a central rod domain. Partial phosphorylation of gfLB3 occurs on Ser-28 in the N-terminal head domain in immature oocytes prior to germinal vesicle breakdown, which suggests continual rearrangement of lamins by a novel lamin kinase in fish oocytes. We applied the expression-screening method to isolate lamin kinases by using phosphorylation site Ser-28-specific monoclonal antibody and a vector encoding substrate peptides from a goldfish ovarian cDNA library. As a result, SRPK1 was screened as a prominent lamin kinase candidate. The gfLB3 has a short stretch of the RS repeats (9-SRASTVRSSRRS-20) upstream of the Ser-28, within the N-terminal head. This stretch of repeats is conserved among fish lamin B3 but is not found in other lamins. In vitro phosphorylation studies and GST-pull down assay revealed that SRPK1 bound to the region of sequential RS repeats (9–20) with affinity and recruited serine into the active site by a grab-and-pull manner. These results indicate SRPK1 may phosphorylate the p34cdc2 site in the N-terminal head of GV-lamin B3 at the RS motifs, which have the general property of aggregation., Highlights • SRPK1 was screened as a prominent lamin kinase candidate from goldfish ovary. • The goldfish lamin B3 (LB3) has RS repeats upstream of the cdc2 target site. • The RS repeats are conserved among fish LB3s but are not found in other lamins. • SRPK1 binds to the RS repeats with affinity and phosphorylates cdc2 site by a grab-and-pull manner.

The jack mackerel Trachurus japonicus is a key commercially exploited fish species in Japan. The rearing experiment often provides information that is useful for understanding the reproductive characteristics of wild stocks; however, there has been no study on spawning in captive T. japonicus. In the study reported here, we induced spawning in T. japonicus caught in the wild by hook and line. Females with fully vitellogenic oocytes and males during spermiation were selected by gonadal biopsy and injected with gonadotropin-releasing hormone analog (GnRHa) mixed in molten coconut butter. This treatment was performed four times in different groups of four females and five to eight males, and each group was maintained in a 3-m3 concrete tank. We observed the first spawning at 1 or 2 days post-injection and collected between 41,690 and 149,450 eggs. Spawning was recorded on 18 consecutive days in one experiment and for 3 days continuously in the other experiments. In the former, spawning ended when the water temperature reached 23 °C and occurred mainly between 2100 and 2400 hours. These results indicate that GnRHa-induced spawning may be useful for evaluating the reproductive characteristics of T. japonicus and obtaining fertilized eggs to conduct larval experiments.

The baculovirus-silkworm expression system is widely used as a mass production system for recombinant secretory proteins. However, the final yields of some recombinant proteins are not sufficient for industrial use. In this study, we focused on the signal peptide as a key factor for improving the efficiency of protein production. Endoplasmic reticulum (ER) translocation of newly synthesized proteins is the first stage of the secretion pathway; therefore, the selection of an efficient signal peptide would lead to the efficient secretion of recombinant proteins. The Drosophila Bip and honeybee melittin signal peptides have often been used in this system, but to the best of our knowledge, there has been no study comparing secretion efficiency between exogenous and endogenous signal peptides. In this study we employed signal peptides from 30K Da and SP2 proteins as endogenous signals, and compared secretion efficiency with those of exogenous or synthetic origins. We have found that the endogenous secretory signal from the 30K Da protein is the most efficient for recombinant secretory protein production in the baculovirus-silkworm expression system.

For multiple-batch-spawning fish species with indeterminate fecundity, income breeding might be more rewarding than capital breeding, because it can fine-tune their reproductive output in terms of maternal condition and offspring survival rate. Although income breeding has been suggested for many small and short-lived pelagic fishes, direct and quantitative evidence for resource allocation to egg production is lacking. This study determined the resource allocation strategy in the Japanese anchovy Engraulis japonicus under ad libitum food availability by using carbon and nitrogen stable isotope ratios (SIRs) as quantitative indicators. Initially, feed with low SIRs was provided and then switched to feed with high SIRs for monitoring the changes in the SIR of eggs during the spawning season. The effect of the latter feed on eggs was evident with the increasing SIRs for both carbon and nitrogen after the diet was switched, suggesting that income resources were primarily used over a season. However, the estimated transfer rates of the resources from food intake to eggs produced varied along with the degree of prevailing temperature. Our findings also suggested that, although well-fed female Japanese anchovy are largely income breeders, the resource allocation strategy used for egg production might be more complicated than previously thought.

The gonadotropins (GtHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are heterodimers composed of a common α subunit (GPα) and a unique β subunit (FSHβ or LHβ); they are synthesized in and secreted from gonadotrophs (FSH and LH cells) in the pituitary. Little is known about the roles of FSH and LH during spermatogenesis in perciform fishes. In this study, we examined immunoreactive changes in FSH and LH cells, and changes in the gene expression of the three gonadotropin subunits in the pituitary of male chub mackerel Scomber japonicus during testicular development. FSHβ-immunoreactive (ir) and LHβ-ir cell area were measured immuno-histochemically based on the FSH and LH cell-occupying area in the proximal pars distalis. The FSHβ-ir cell area increased significantly during spermiation, while FSHβ mRNA levels, already high at the beginning of spermatogenesis, increased further, peaking during spermiation. In contrast, LHβ-ir cell area and LHβ mRNA levels, which were low at the beginning of spermatogenesis, increased significantly during late spermatogenesis, peaking during spermiation. For both FSH and LH, GtHβ-ir cell area and GtHβ mRNA levels decreased until gonadal resting. GPα mRNA levels showed similar changes to LHβ mRNA levels. These results suggest that in the chub mackerel, FSH may play an important role in the early and late phases of spermatogenesis, and that LH may play a role during late spermatogenesis and spermiation. Moreover, our results demonstrate that changes in GtHβ-ir cell area were accompanied by similar changes in the expression of the FSHβ and LHβ genes, both of which increased during testicular development.

Sex steroids have been suggested to be involved in gonadal sex change in hermaphrodite fish. Aromatase, the enzyme that catalyzes the conversion of androgens into estrogens, is a principal enzyme regulating gonadal sex. However, the detailed functions of each steroid hormone remain to be evaluated. Recent studies have demonstrated that estradiol-17β (E2) is synthesized via estrone (E1) in some hermaphrodite species. On the other hand, 11-ketotestosterone (11KT) is produced in the testis via testosterone (T). In this study, we hypothesized that E1 and T are also involved in the sex change as precursors for E2 and 11KT, respectively. We implanted females of the wrasse, Pseudolabrus sieboldi, with T and 11KT, and males with E1 and E2, by use of sustained-release capsules. In females, testicular tissues and body color change were observed after androgen administration, in which 11KT was more effective than T. In contrast, after estrogen administration, the gonads of males contained oocytes. In females, the administration of T and 11KT resulted in reduced serum E2 levels. Conversely, serum 11KT levels decreased in the E1- and E2-treated males. Thus, we successfully induced bidirectional sex change in the gonad by estrogen and androgen administration in vivo. Moreover, this study raises the possibility that E1 and T are involved in the sex change as precursors for E2 and 11KT, respectively. J. Exp. Zool. 317A:552–560, 2012. © 2012 Wiley Periodicals, Inc.

Gonadotropin-releasing hormone (GnRH) is a key neuroendocrine peptide involved in the reproduction of fish and other vertebrates. However, characterizing the involvement of GnRH in fish reproduction has been complicated by the discovery of multiple GnRH forms. In the present study, we isolated full-length cDNAs encoding three GnRH forms and analyzed seasonal changes in the concentrations of mRNA in the brain and corresponding peptides in the brain and pituitary, in relation to seasonal gonadal development of chub mackerel (Scomber japonicus). Chub mackerel sbGnRH, cGnRH-II, and sGnRH cDNAs encode 98, 85, and 90 deduced amino acids, respectively. In females, brain sbGnRH mRNA and peptide concentrations were significantly higher only during the post-spawning season (August); however, pituitary peptide concentrations were higher during late vitellogenesis (April) and the post-spawning season, in comparison to immature stage (November). In males, brain sbGnRH mRNA and pituitary peptide concentrations were higher during spermiation (April). No significant differences in cGnRH-II mRNA or peptide concentrations were found in either sex. Furthermore, in females, brain sGnRH mRNA concentrations did not vary significantly; however, corresponding peptide concentrations in the brain and pituitary were higher during late vitellogenesis and the post-spawning season, respectively. In males, only brain sGnRH mRNA concentrations were higher during the post-spawning season, with no significant change in peptide concentrations. This study quantified the seasonal expression changes of three GnRH mRNAs and peptides in both sexes of chub mackerel, and the present results combined with our previous immunocytochemical report indicates that sbGnRH form plays a dominant role in seasonal gonadal development.