Localization of leptin and leptin receptor in the bovine adenohypophysis

The present study was carried out to detail the cellular localization of leptin (Lep) and the leptin receptor (LepR) in the bovine adenohypophysis. Lep immunoreactivity (Lep-ir) was found in about 30% of adenohypophysial cells in the gland. Immunochemistry of Lep and specific hormones using serial sections revealed that Lep-ir was present in 60.4% of somatotrophs, 15.9% of gonadotrophs, 6.5% of mammotrophs, 6.5% of thyrotrophs and 2.4% of corticotrophs. Both the common short isoform (OBRa) and the long isoform (OBRb) of LepR mRNA were expressed in the bovine adenohypophysis. LepR immunoreactivity (LepR-ir) was found in only 2.8% of the adenohypophysial cells and over 50% of LepR-ir cells were gonadotrophs, in which most of the cells were distributed in the zona tuberalis. The findings on Lep and LepR in the adenohypophysial cells indicate that Lep may regulate gonadotroph function through autocrine/paracrine pathway in the bovine adenohypophysis.     

Isolation and culture of rabbit primordial germ cells

Primordial germ cells (PGCs) are embryonic precursors of the gametes of adult animals and are considered stem cells of the germline. Since their proliferation in vitro correlates well with the schedule of developmental changes in vivo, they might be interesting research tools for genomic imprinting, germ-cell tumors and fertility. Furthermore, once primordial germ cells are separated and placed on a feeder layer with cytokines, they become cultured pluripotent cell lines called embryonic germ (EG) cells. EG cells share several important characteristics with embryonic stem (ES) cells as they can also contribute to the germ line of chimeras. To investigate the characteristics of PGCs and establish rabbit EG (rEG) cells, we cultured rabbit PGCs (rPGCs) in vitro with various combinations of leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and forskolin on inactivated mouse embryonic fibroblast (MEF) feeder layers. The present study found PGC proliferation in early cultures and induction of rEG-like colonies. These cells expressed pluripotent markers, such as alkaline phosphatase activity, OCT-4, Sox-2 and SSEA-1, in the undifferentiated state; however, the cells did not develop into a teratoma when injected into the kidney capsules of SCID mice, although the restricted differentiation potentials to neural cells were determined via embryoid body formation. From these characteristics and further characterization of the germ stem cell markers Vasa, SCP-1 and SCP-3, we suggested that these were hybrid cells with characteristics somewhere between PGC and EG cells.     

Phenotypic and functional analysis of bovine peripheral blood dendritic cells before parturition by a novel purification method

Dendritic cells (DCs) are specialized antigen presenting cells specializing in antigen uptake and processing, and play an important role in the innate and adaptive immune response. A subset of bovine peripheral blood DCs was identified as CD172a⁺/CD11c⁺/MHC (major histocompatibility complex) class II⁺ cells. Although DCs are identified at 0.1%–0.7% of peripheral blood mononuclear cells (PBMC), the phenotype and function of DCs remain poorly understood with regard to maintaining tolerance during the pregnancy. All cattle used in this study were 1 month before parturition. We have established a novel method for the purification of DCs from PBMC using magnetic‐activated cell sorting, and purified the CD172a⁺/CD11c⁺ DCs, with high expression of MHC class II and CD40, at 84.8% purity. There were individual differences in the expressions of CD205 and co‐stimulatory molecules CD80 and CD86 on DCs. There were positive correlations between expression of cytokine and co‐stimulatory molecules in DCs, and the DCs maintained their immune tolerance, evidenced by their low expressions of the co‐stimulatory molecules and cytokine production. These results suggest that before parturition a half of DCs may be immature and tend to maintain tolerance based on the low cytokine production, and the other DCs with high co‐stimulatory molecules may already have the ability of modulating the T‐cell linage.     

Management and yield prediction of Kunugi (Quercus acutissima) grazing forests

This study deals with grazing in kunugi (Quercus acutissima) forests in the Aso district of Kyushu Island in southwest Japan. These forests are managed for production of bed-logs for shiitake mushrooms and cow-calf farming. One of their characteristics is short-term rotation such as 10–15 years for bed-logs and a year for calf production. A forest grazing experiment was begun in Minamioguni to look at forest growth, vegetation change and grazing intensity. Stem densities dropped in a few years. After sprout cutting, they also dropped gradually, then stabilized. Although grazing caused tree damage and suppressed tree growth, grazing intensity of up to 150 cow-days/ha·year did not harm forest regeneration. Herbage volume decreased as grazing was repeated and trees grew. Another investigation of kunugi grazing forests in Minamioguni and Asaji showed the correlation betweenRy (yield index in Stand density diagram) and grazing capacity could be expressed with a regression equation. The results were also used to design a yield table for kunugi grazing forests. The yield table has items ofRy and grazing capacity in addition to usual yield table items, and can indicate timber yield and grazing capacity at the same time. The table estimates that proper grazing capacity is 60–80 cow-days/ha·year in wild grass sites. In the light of these results, an optimal management plan was proposed as a diagram integrating stem density, forest yield, and forest management.     

Linkage analysis of RFLP markers for clubroot resistance and pigmentation in Chinese cabbage (Brassica rapa ssp. pekinensis)

A restriction fragment length polymorphism (RFLP) – based linkage map of Chinese cabbage (Brassica rapa ssp. pekinensis) (2n=20) including two agronomic traits, clubroot resistance and orange-yellow pigmentation, was constructed using doubled haploid parents. The total linkage distance was 735 cM; 63 loci were distributed into ten linkage groups. Clubroot resistance of the parental line T136-8 to the current pathotype, race 2, was predominantly controlled by a single dominant gene that originated from European turnip. The locus for clubroot resistance by the dominant major gene (CRa) was mapped on linkage group 3, and RFLP loci HC352b and HC181 were located 3 cM and 12 cM from it, respectively. The locus HC352b was identified by a 4.4 Kb Eco R I fragment, which segregated for null allele. The absence of an allelic fragment in HC352b could be interpreted by deletion in the resistance source; homozygotes for CRa could be efficiently selected by detecting null types for the marker. Orange-yellow pigmentation expressed in head inner leaves and petals was governed by a single recessive gene. The locus (Oy) for the pigmentation was mapped on linkage group 1, being located 17–19 cM from three RFLP loci that were closely linked to each other. The linkage analysis for clubroot resistance and unique pigmentation revealed some informative RFLP markers. Identification of molecular markers for clubroot resistance and other agronomically important traits would provide useful information in breeding programs of Chinese cabbage. This revised version was published online in August 2006 with corrections to the Cover Date.     

Antibodies produced by mice immunized with recombinant vaccinia viruses expressing two different types of a major Theileria sergenti surface antigen (p32) react with the native surface antigen.

A 32 kDa major surface antigen, p32, of Theileria sergenti at the piroplasm stage is the main target of the host immune response. The immunogenic property of the p32 varies in some strains among the population of Theileria sergenti in Japan where the Chitose type and the Ikeda type are the most common varieties. We have constructed vaccinia virus recombinants vv/p32C and vv/p32I which harbor the Chitose and Ikeda types of p32 gene, respectively. It was found that vv/p32C and vv/p32I produced type-specific p32 which did not cross react with the monoclonal antibodies (mAbs) against the other type of p32. When mice were immunized with vv/p32C and vv/p32I, antibodies against p32 were detectable 2 weeks after the immunization, and these antibodies reacted with the native surface antigen in purified T. sergenti merozoite.     

Production of androgenetic amago salmon<Emphasis Type="Italic"> Oncorhynchus masou ishikawae</Emphasis> with dispermy fertilization

With the aim of improving artificial androgenesis in teleost fishes, we tested two methods for producing androgenetic diploids of amago salmon (Oncorhynchus masou ishikawae), namely, fertilization of gamma-ray irradiated eggs with fused spermatozoa (sperm-fusion method) and the fertilization of irradiated eggs with untreated sperm followed by the blocking of cell division (mitosis-inhibition method). Our results showed that the optimal condition for sperm fusion was to treat the sperm with 50% polyethylene glycol (molecular weight 7500) for 100 s. The efficiency of the two methods of androgenesis was compared in terms of fertilization rate, hatching rate, and larval survival after hatching. The rate of fertilization was lower with the sperm-fusion method than with the mitosis-inhibition method, but the reverse was true for the hatching rate. The survival rate of hatched larvae was the same with the two methods. Androgenesis was confirmed with a recessive albino color marker, and all viable offspring were found to be heterozygous based on analysis of the microsatellite markers. Our results suggest that androgenesis with the sperm-fusion method is a promising approach with potential applications in both aquaculture breeding programs and the preservation of endangered freshwater fishes.     

Examination of a practical method for zinc enrichment of euryhaline rotifers (Brachionus plicatilis)

Larval growth and survival of marine finfish in mass seed production are affected by the nutritional value of live feeds such as rotifers and Artemia. Thus far, many studies have been conducted to develop effective methods for the enrichment of live feeds with essential fatty acids and vitamins. In this study, a practical method for enrichment of rotifers with zinc was investigated. Changes in the concentrations of other minerals when zinc was added to the rotifer-enrichment tanks were also studied. The mineral composition of rotifers and Chlorella after zinc enrichment revealed that the direct addition of zinc to the culture media was not effective because rotifers cannot efficiently accumulate waterborne zinc. The ability of Chlorella to absorb waterborne zinc is much higher than that of rotifers, and hence, zinc was pre-accumulated in Chlorella, which was then fed to the rotifers. The maximum zinc content of the rotifers was 585.0 μg g^? 1 (dry matter) when the rotifers were enriched with zinc alone. This zinc concentration is comparable to that found in natural zooplankton. In rotifers simultaneously enriched with zinc and n?3 highly unsaturated fatty acids (HUFAs), the zinc content increased, but the n?3 HUFA content did not. Therefore, separate enrichment with zinc and fatty acids was adopted. The zinc content of rotifers fed zinc-enriched Chlorella was significantly higher than that of rotifers fed unenriched Chlorella. After zinc enrichment, rotifers were enriched with fatty acids, and the docosahexaenoic acid (DHA) and n?3 HUFA levels in rotifers were higher than the levels obtained after simultaneous enrichment with zinc and fatty acids. With regard to the concentration of other minerals in rotifers after zinc enrichment, the manganese content tended to decrease when the zinc content increased.The results of this study demonstrated that zinc enrichment of rotifers was successfully performed by using microalgae that had accumulated zinc, and the enrichment of rotifers with fatty acids was also achieved after the completion of zinc enrichment and before feeding the larvae. This method could be utilized for the enrichment of zooplankton with other minerals as well.     

Amberjack <Emphasis Type="Italic">Seriola dumerili</Emphasis> interleukin-10 negatively suppresses host cell-mediated immunity

Interleukin-10 (IL-10) is an anti-inflammatory cytokine and negatively regulates cell-mediated immunity (CMI) induction by inhibiting cytokine production in type 1 T helper cells. IL-10 genes have been isolated from several fish, and inflammatory cytokine inhibition by IL-10 has been well examined. However, a CMI regulator of IL-10 in fish has not yet been identified. In this study, we cloned the IL-10 gene in amberjack Seriola dumerili and analyzed its function using its recombinant protein (rIL-10). In an in vitro culture experiment, gene expression of inflammatory cytokines was suppressed in leukocytes incubated with rIL-10 compared with cells that only received Nocardia seriolae stimulation. This result suggests amberjack IL-10 has conserved function as an inflammatory cytokine inhibitor. Bactericidal activity of amberjack cells against intracellular pathogen stimulation was decreased in a rIL-10 dose-dependent manner. Furthermore, a significant reduction in the T-bet/GATA-3 ratio was observed in N. seriolae living cell (LC)?+?rIL-10-injected fish. Taken together, these results suggest amberjack rIL-10 suppresses CMI induction both in vitro and in vivo. In addition, the number of IgM⁺ cells among spleen leukocytes in N. seriolae?+?rIL-10-injected fish was higher than in only N. seriolae LC, suggesting that Th2-dominant immunity was induced by adding rIL-10.