Effects of dietary silkrose of Antheraea yamamai on gene expression profiling and disease resistance to Edwardsiella tarda in Japanese medaka (Oryzias latipes).

We previously identified a novel acidic polysaccharide, silkrose-AY, from the Japanese oak silkmoth (Antheraea yamamai), which can activate an innate immune response in mouse macrophage cells. However, innate immune responses stimulated by silkrose-AY in teleosts remain unclear. Here, we show the influence of dietary silkrose-AY in medaka (Oryzias latipes), a teleost model, in response to Edwardsiella tarda infection. Dietary silkrose-AY significantly improved the survival of fish and decreased the number of bacteria in their kidneys after the fish were artificially infected with E. tarda by immersion. We also performed a microarray analysis of the intestine, which serves as a primary barrier against microbial infection, to understand the profiles of differentially expressed genes (DEGs) evoked by silkrose-AY. The dietary silkrose-AY group showed differential expression of 2930 genes when compared with the control group prior to E. tarda infection. Gene ontology and pathway analysis of the DEGs highlighted several putative genes involved in pathogen attachment/recognition, the complement and coagulation cascade, antimicrobial peptides/enzymes, opsonization/phagocytosis, and epithelial junctional modification. Our findings thus provide fundamental information to help understand the molecular mechanism of bacterial protection offered by insect-derived immunostimulatory polysaccharides in teleosts.

Sex-changing patterns of Akoya pearl oyster (Pinctada fucata).

BACKGROUND: Pearl production by transplantation in Akoya pearl oyster (Pinctada fucata) is a biotechnology developed in Japan that skillfully utilizes the pearl-forming ability of oysters. In this method, cultured pearls are formed from a pearl nucleus and a small piece of mantle transplanted into the gonads of recipient pearl oysters. In this study, we hypothesized that the sex of the recipient pearl oyster might affect the quality of pearl produced. While some previous studies have examined the sex of Akoya pearl oyster, detailed information is lacking. RESULTS: To investigate sex in Akoya pearl oyster, we collected small gonadal fragments from 1-year-old pearl oysters by biopsy. Using the collected gonad fragment, the sex of the oysters was determined by microscopic observation, and the remaining samples were stored for gene expression analyses. All oysters were labeled to distinguish each individual for serial samplings every four months over the 2-year study period. At the start of experiment, nearly all of the pearl oysters were male, but the male:female ratio ofmale decreased over the course of the experiment. Interestingly, the number of males increased after spring, during the breeding season. This suggests that, in pearl oyster, sex is affected by season. Expression analysis of sex-related genes (Dmrt2, Vtg, Zp) indicated that all genes were expressed in all individuals and all periods. CONCLUSIONS: These results suggest that Akoya pearl oysters are hermaphroditic, and that females appear as necessary, such as during the breeding season. These findings could contribute to higher efficiency and quality of pearl cultivation.

Effect of the squid viscera hydrolysate on growth performance and digestion in the red sea bream Pagrus major.

The improvement in feed efficiency is one of the most important subjects in fish culture. The development of feed, in terms of good intake, high growth performance, and high feed efficiency is needed. Squid viscera are one of the candidates for alternative material in improving feed efficiency in fish culture. In the present study, we described the dietary effect of the squid viscera hydrolysate (SVH) on the growth performance of the red sea bream. The addition of SVH to feed caused significant increases in feed intake, fork length, and body weight and produced a marked improvement in feed conversion after 4 weeks of feeding. Furthermore, the results of this feeding revealed that low dietary levels of SVH promote growth performance in the red sea bream. We physiologically analyzed digestion and appetite in fish fed diet containing SVH. SVH promoted the activity of hepatic trypsin and lipase, gene expression of stomach pepsin, hepatic lipase, and pyloric caeca trypsin, thereby improving the nutrient availability in red sea bream. Moreover, the mRNA expression of appetite regulating factor, such as brain NPY and stomach ghrelin was significantly improved by dietary SVH. Our current results indicate that dietary SVH as alternative material produced excellent effects on growth performance, which is dependent on the promoting effect on digestion and appetite in red sea bream.

Surface-spreading technique of meiotic cells and immunodetection of synaptonemal complex proteins in teleostean fishes.

BACKGROUND: Different moderrn methodologies are presently available to analyze meiotic chromosomes. These methods permit investigation of the behavior of chromosomes in the normal complement and of sex and B chromosomes, two special types of chromosomes that are associated with the A complement and are present in many organisms, including fishes. However, meiotic studies are still scarce in fishes, considering the wide number of species in this group.. Here, we describe a new protocol for the visualization of the synaptonemal complex in spermatocytes and oocytes of fishes and to the sequential use of the technique with other procedures and techniques such as immunodetection of the synaptonemal complex protein with a specific antibody and co-detection of DNA sequences by FISH. RESULTS: The meiotic surface-spreading protocol used in the present proposal worked well in representative species of four fish orders and was useful in obtaining good results even in small specimens. Fish-specific antibodies and commercial products worked similarly well to detect synaptonemal complex (SC) proteins. The sequential application of fluorescence in situ hybridization using specific probes showed clear signals associated with the SC structures identified by immunostaining. CONCLUSION: Here, we provide a useful and applicable immunofluorescent protocol for the visualization of synaptonemal complex proteins in the meiotic cells of fishes in surface-spreading preparations. Furthermore, this technique allows for the sequential application of other cytogenetic procedures.

A xenograft mantle transplantation technique for producing a novel pearl in an akoya oyster host.

The brightness and color of pearls varies among different pearl-producing shellfish and have been a source of human fascination since ancient times. When produced through cultivation, the characteristics and quality of a pearl depend on the kind of shellfish used and also the transplanted mantle graft. This suggests that the Akoya pearl oyster, which is generally used in Japan for pearl culturing, can produce different kinds of pearl through the use of mantles from different species of shellfish. However, a transplanted heterogeneous mantle would be rejected by the immune system of the Akoya oyster. We have therefore developed a new method to suppress the Akoya immune system that archives immune tolerance to other shellfish. It is generally known that small quantities of antigens can be used to produce archived immunological tolerance in a clinical setting. We successfully suppressed the Akoya pearl oyster immune response against a Mabé pearl oyster graft through repeat injections of mantle homogenates. We then transplanted a Mabé pearl oyster mantle graft into the immunologically tolerant Akoya pearl oyster and obtained a Mabé pearl from an Akoya pearl oyster. Our new technique thus makes the production of novel and different pearls in the Akoya possible. We believe that this has significant future potential for the advancement of the pearl industry.

Trypsin regulates meiotic initiation in the Japanese eel (Anguilla japonica) by promoting the uptake of taurine into germ cells during spermatogenesis.

Meiosis is a unique and critical process in reproduction. Although the key molecular components of meiosis have been identified, the molecular mechanisms regulating the entry into this pathway remain unclear. We previously demonstrated that a progestin in teleost fish, 17alpha, 20beta-dihydroxy-4-pregnen-3-one, is essential for meiotic initiation, and up-regulates taurine synthesis and the production of trypsin in Sertoli cells. In the present study, we found that trypsin promotes the uptake of taurine into germ cells through the up-regulation of solute carrier family 6 (neurotransmitter transporter, taurine), member 6 (Slc6a6) expression. We further found that this up-regulation of the taurine signal is required for Spo11a expression and meiotic initiation.

Gonads directly regulate growth in teleosts.

In general, there is a relationship between growth and reproduction, and gonads are known to be important organs for growth, but direct evidence for their role is lacking. Here, using a fish model, we report direct evidence that gonads are endocrine organs equal to the pituitary in controlling body growth. Gonadal loss of function, gain of function, and rescue of growth were investigated in tilapia. Gonadectomy experiments were carried out in juvenile males and females. Gonadectomy significantly retarded growth compared with controls; however, this retardation was rescued by the implantation of extirpated gonads. Because gonads express growth hormone, it is possible that gonads control body growth through the secretion of growth hormone and/or other endocrine factors. We propose that gonads are integral players in the dynamic regulation of growth in teleosts.

Tolerance of spermatogonia to oxidative stress is due to high levels of Zn and Cu/Zn superoxide dismutase.

BACKGROUND: Spermatogonia are highly tolerant to reactive oxygen species (ROS) attack while advanced-stage germ cells such as spermatozoa are much more susceptible, but the precise reason for this variation in ROS tolerance remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: Using the Japanese eel testicular culture system that enables a complete spermatogenesis in vitro, we report that advanced-stage germ cells undergo intense apoptosis and exhibit strong signal for 8-hydroxy-2'-deoxyguanosine, an oxidative DNA damage marker, upon exposure to hypoxanthine-generated ROS while spermatogonia remain unaltered. Activity assay of antioxidant enzyme, superoxide dismutase (SOD) and Western blot analysis using an anti-Copper/Zinc (Cu/Zn) SOD antibody showed a high SOD activity and Cu/Zn SOD protein concentration during early spermatogenesis. Immunohistochemistry showed a strong expression for Cu/Zn SOD in spermatogonia but weak expression in advanced-stage germ cells. Zn deficiency reduced activity of the recombinant eel Cu/Zn SOD protein. Cu/Zn SOD siRNA decreased Cu/Zn SOD expression in spermatogonia and led to increased oxidative damage. CONCLUSIONS/SIGNIFICANCE: These data indicate that the presence of high levels of Cu/Zn SOD and Zn render spermatogonia resistant to ROS, and consequently protected from oxidative stress. These findings provide the biochemical basis for the high tolerance of spermatogonia to oxidative stress.

Production of transgenic medaka fish carrying fluorescent nuclei and chromosomes.

As with zebrafish, attention has focused on the teleost medaka Oryzias latipes as an experimental animal representative of non-mammalian vertebrates in various fields of biological science. To enable real-time analyses of the dynamics of nuclei and chromosomes in living medaka cells, we produced a transgenic medaka expressing a fusion protein between histone H2B and green fluorescent protein (GFP) under the control of a cytomegalovirus (CMV) promoter. Since the nuclei and chromosomes of transgenic medaka cells are labeled with GFP, their morphological changes can be instantly monitored throughout the mitotic cell cycle progression under a fluorescent microscope without any fixation and staining of samples. However, GFP-labeling of nuclei and chromosomes is not successful during early embryonic development until zygotic expression begins and during the meiotic cell cycle progression, because the CMV promoter does not work in these stages. In addition, histone H2B-GFP fusion proteins are expressed in an organ-specific manner; strong and ubiquitous expression occurs in cells comprising the gut and fin, whereas the expression is restricted to certain types of cells in the liver and brain. These findings suggest that the CMV-driven expression of the histone H2B-GFP transgene is modified depending on the integration site of the transgene in the genome. Nevertheless, easy and precise monitoring of cytological changes in nuclei and chromosomes in the majority of mitotic cells by using the transgenic medaka will greatly contribute to a better understanding of control mechanisms of nuclear and chromosomal behaviors in vertebrate cells.

Artificial fertilization by intracytoplasmic sperm injection in a teleost fish, the medaka (Oryzias latipes).

Intracytoplasmic sperm injection (ICSI) is a technique that has been successfully used for assisting reproduction in mammals. However, this method is still not reliable in nonmammalian species, including teleosts. We succeeded in producing medaka individuals by ICSI with a rate of 13.4% (28 hatched embryos out of 209 eggs fertilized by ICSI), the best value reported so far in teleosts, including zebrafish and Nile tilapia. Although the technique was based on that developed for mammalian eggs, some critical modifications were made to adjust it to the medaka egg, which has a thick and hard envelope (the chorion) and a single sperm entry site (the micropyle). Medaka ICSI was performed by injecting a demembranated spermatozoon into an egg cytoplasm through the micropyle 10-15 sec after egg activation induced by a piezo-actuated vibration, the site and timing of sperm penetration being consistent with those in normal fertilization in medaka. To increase the efficiency of ICSI in medaka, we found that the fertilization by ICSI should precisely mimic the fertilization by insemination with intact sperm, both spatially and temporally. The success rate of ICSI was highly variable in batches of eggs (ranging from 0% to 56%), suggesting that the conditions of eggs are important factors in stabilizing the production of individuals by ICSI. The success in medaka ICSI provides a basis for future research to understand the basic mechanisms in gamete biology of teleosts as well as for development of new technology that can yield valuable applications in fisheries science.

Identification and expression analysis of rainbow trout pumilio-1 and pumilio-2.

Pumilio is a sequence-specific RNA-binding protein that regulates translation from the relevant mRNA. The PUF-domain, the RNA-binding motif of Pumilio, is highly conserved across species. In the present study, we have identified two pumilio genes (pumilio-1 and pumilio-2) in rainbow trout and analyzed their expression patterns in its tissues. Pumilio-1 mRNA and pumilio-2A mRNA code for typical full length Pumilio proteins that contain a PUF-domain, whereas pumilio-2B mRNA is a splice variant of pumilio-2 and encodes a protein that lacks the PUF-domain. We have also identified a novel 72-bp exon that has not been reported in other animal species but is conserved in fish species. The insertion of this novel exon leads to the expression of an isoform of the Pumilio-2 protein with a slightly altered conformation of the PUF-domain. Pumilio-1 mRNA and pumilio-2A mRNA (irrespective of the presence of the 72-bp exon) are expressed in both the brain and ovaries at high levels, whereas pumilio-2B mRNA is expressed at low levels in all the rainbow trout tissues examined. Western blot analysis also indicates that the full length Pumilio proteins are expressed predominantly in the brain and ovaries. These data suggest that the Pumilio proteins have physiological roles and are involved in regulatory mechanisms in rainbow trout.

Structural components of the synaptonemal complex, SYCP1 and SYCP3, in the medaka fish Oryzias latipes.

The synaptonemal complex (SC) is a meiosis-specific structure essential for synapsis of homologous chromosomes. For the first time in any non-mammalian vertebrates, we have isolated cDNA clones encoding two structural components of the SC, SYCP1 and SYCP3, in the medaka, and investigated their protein expression during gametogenesis. As in the case of mammals, medaka SYCP1 and SYCP3 are expressed solely in meiotically dividing cells. In the diplotene stage, SYCP1 is diminished at desynaptic regions of chromosomes and completely lost on the chromosomes at later stages. SYCP3 is localized along the arm and centromeric regions of chromosomes at metaphase I, and its existence on the whole chromosomes persists up to anaphase I, a situation different from that reported in the mouse, in which SYCP3 is confined to the centromeric regions but lost on the arm regions at metaphase I. Thus, the expression patterns of SC components are different in mammals and fish despite the resemblance in morphological structure of the SC, suggesting divergence in the function of the SC in regulation of meiosis-specific chromosomal behavior. Since the antibody against medaka SYCP3 is cross-reactive to other fishes, it should be generally useful for a meiosis-specific marker in fish germ cells.

Spatio-temporal expression of a DAZ-like gene in the Japanese newt Cynops pyrrhogaster that has no germ plasm.

To investigate the germ cell specification in urodeles, we cloned a DAZ-like sequence from the Japanese newt Cynops pyrrhogaster, Cydazl, and raised antibodies specific to Cydazl. Cydazl is a homologue of the human DAZ (deleted in azoospermia), DAZL, and Xenopus dazl genes, which are involved in gametogenesis or germ cell specification. During gametogenesis, expression of Cydazl mRNA and Cydazl protein was detected at first in the small previtellogenic oocytes in females but was not localized as seen in Xenopus and was restricted to secondary spermatogonia prior to meiosis in males. During early embryogenesis, maternal stores of the Cydazl transcript and protein were present in the entire embryos, not localized in any specific region. The zygotic expression was detected in hatching larvae (stage 50) by RT-PCR analysis whereas specific cells expressing Cydazl could not be determined by in situ hybridization at this stage. Strong expression of Cydazl and Cydazl were detected in primordial germ cells (PGCs) that had entered the gonadal rudiment at late stage 59. These results suggest that Cydazl does not function early in development, for the specification of germ cells, but functions later for differentiation of germ cells in the developing gonads during embryogenesis and for meiotic regulation, supporting the previous idea of an intermediate germ cell formation mode in urodeles.

Changes in the expression and localization of cohesin subunits during meiosis in a non-mammalian vertebrate, the medaka fish.

Until the onset of anaphase, sister chromatids are bound to each other by a multi-subunit protein complex called cohesin. Since chromosomes in meiosis behave differently from those in mitosis, the cohesion and separation of homologous chromosomes and sister chromatids in meiosis are thought to be regulated by meiosis-specific cohesin subunits. Actually, several meiosis-specific cohesin subunits, including Rec8, STAG3 and SMC1beta, are known to exist in mammals; however, there are no reports of meiosis-specific cohesin subunits in other vertebrates. To investigate the protein expression and localization of cohesin subunits during meiosis in non-mammalian species, we isolated cDNA clones encoding SMC1alpha, SMC1beta, SMC3 and Rad21 in the medaka and produced antibodies against recombinant proteins. Medaka SMC1beta was expressed solely in gonads, while SMC1alpha, SMC3 and Rad21 were also expressed in other organs and in cultured cells. SMC1beta forms a complex with SMC3 but not with Rad21, in contrast to SMC1alpha, which forms complexes with both SMC3 and Rad21. SMC1alpha and Rad21 were mainly expressed in mitotically dividing cells in the testis (somatic cells and spermatogonia), although their weak expression was detected in pre-leptotene spermatocytes. SMC1beta was expressed in spermatogonia and spermatocytes. SMC1beta was localized along the chromosomal arms as well as on the centromeres in meiotic prophase I, and its existence on the chromosomes persisted up to metaphase II, a situation different from that reported in the mouse, in which SMC1beta is lost from the chromosome arms in late pachytene despite its universal presence in vertebrates.

Temporally and spatially selective loss of Rec8 protein from meiotic chromosomes during mammalian meiosis.

Sister chromatid cohesion is maintained from DNA replication to metaphase-to-anaphase transition by multisubunit protein complexes called cohesin, which include at least four proteins, SMC1alpha, SMC3, Rad21 and either SA1 or SA2, in mammalian somatic cells. We report here the first evidence of the involvement of Rec8 protein, a mammalian homolog of yeast Rec8p, in meiosis-specific chromosome behavior in mammals. In immunoblotting and immunohistochemical analysis using specific antibodies against mouse Rec8, we found that Rec8 was expressed in the testis but not in the kidney or liver; more precisely, it was expressed in spermatocytes and spermatids but not in spermatogonia or other somatic cells. We also found that Rec8 is present in both phosphorylated and dephosphorylated states in vivo. Immunoprecipitation analyses revealed that Rec8 associates with other cohesin proteins, SMC1beta (meiosis-specific protein) and SMC3 and with a component of synaptonemal complexes, SCP3, but not with SMC1alpha. In meiotic chromosome spreads, Rec8 was localized along the axial/lateral elements of the synaptonemal complexes in meiotic prophase from the leptotene to diplotene stages. At later stages, diakinesis and metaphase I, Rec8 was localized along the interstitial axes of chromosomes, including both centromere and arm regions of chromosomes. However, concomitantly with separation of homologous chromosomes in anaphase I, Rec8 was no longer detected along the arm regions, while it persisted on centromere regions up to metaphase II. In anaphase II, the centromeric signals were diminished. We propose from these results that mammalian Rec8 protein, in association with SMC3 and SMC1beta but not SMC1alpha, is involved in meiosis-specific chromosome behavior, and that homologous chromosome separation is triggered by selective loss of Rec8 from chromosome arms in meiosis I, while sister chromatid cohesion is maintained until metaphase II/anaphase II transition by centromeric Rec8 during mammalian meiosis.