Production of inhibin A and inhibin B in boars: changes in testicular and circulating levels of dimeric inhibins and characterization of inhibin forms during testis growth

We investigated the production of inhibin in boars from the infantile to pubertal periods by: (1) measurement of testicular and circulating levels of inhibin, (2) characterization of inhibin forms and (3) localization of inhibin subunits in the testis. Total inhibin levels in the testis increased until 8 weeks of age but then declined to much lower values at 15 weeks. Testicular inhibin A and inhibin B were high until 8 weeks. Circulating levels of total inhibin and inhibin A were also high until 8 weeks, then declined from 10 weeks; inhibin B was not detected, because of low sensitivity of the inhibin B assay. Analyses of inhibin A and inhibin B levels in the eluted fractions obtained from testes after immunoaffinity chromatography and SDS-PAGE showed the presence of a peak of approximately 45 kDa until 10 weeks of age. As the boars aged, the levels of inhibin A and inhibin B increased in the molecular weight region of 29–31 kDa. The fractions corresponding to 29 and 30 kDa suppressed FSH release from rat pituitary cells, but the 45 kDa fraction had no FSH-suppressing activity. Total amounts of inhibin A isolated from the SDS gels were similar to those of inhibin B until 10 weeks of age, but were three times higher than those of inhibin B between 15 and 25 weeks. Further fractionation by reverse phase high-performance liquid chromatography revealed that the 29–31 kDa immunoreactive material was composed of mature forms of inhibin A and inhibin B, in addition to a 26 kDa monomer. Immunohistochemistry indicated that positive immunostaining for the subunits was observed in Sertoli cells from the infantile to pubertal periods. Elongated spermatids also showed positive signals at age 25 weeks. These results clearly indicated that: (1) the boar testis has the ability to produce inhibin A and inhibin B during the infantile period but inhibin A is the predominant form towards puberty and (2) the molecular weight forms of inhibin and the sites of production of inhibin change with testicular development.     

Effect of Wheat Starch Structure on Swelling Power

Starches were isolated from the endosperm of 12 wheat samples with a wide swelling power range in the wholemeal. Starch amylose content (24.8–34.2%) correlated negatively with the swelling power of isolated starch (18.3–26.9), but starch lipid content showed no such correlation. Higher proportions of long chains (DP ≥ 35) in amylopectins contributed to increased starch swelling. Native starch gelatinization temperatures and enthalpy measured by differential scanning calorimetry correlated positively with swelling power, which also correlated significantly with the regelatinization enthalpy of retrograded starches stored at 5°C for two and four weeks.     

Nursery habitat use for larval and juvenile Pacific herring Clupea pallasii in Miyako Bay on the Pacific coast of northern Japan

Fisheries Science - To clarify nursery habitat use for larval and juvenile Pacific herring Clupea pallasii, we collected eggs, larvae, and juveniles at several sampling sites in inner Miyako Bay...     

Identification of QTLs controlling rice drought tolerance at seedling stage in hydroponic culture

‘Drought avoidance’ and ‘drought tolerance’ are two mechanisms by which plants adapt under water stress. These mechanisms are difficult to evaluate separately in field experiments. Using hydroponic culture, we studied the genetic control of drought tolerance in rice (Oryza sativa L.) without the effect of drought avoidance. A backcross inbred population of ‘Akihikari’ (lowland cultivar) × ‘IRAT109’ (upland cultivar) with 106 lines was cultured with (stressed condition) and without (non-stressed condition) polyethylene glycol (PEG) at seedling stage. The relative growth rate (RGR), specific water use (SWU), and water use efficiency (WUE) showed significant genotype × environment interactions with or without PEG, indicating that each line responded differently to water stress. A quantitative trait locus (QTL) analysis revealed that these interactions were QTL specific. A total of three QTLs on chromosomes 2, 4, and 7 were detected for RGR. The QTL on chromosome 7 had a constant effect across environments, while the QTL on chromosome 4 had an effect only under non-stressed condition and that on chromosome 2 only under stressed condition. The stress-specific QTL on chromosome 2 was not co-located with any QTLs for root system depth previously reported from the same mapping population. However, this QTL was co-located with a stress-specific QTL for SWU, suggesting that the control of transpiration was relevant to dry matter production under drought. We concluded that PEG-treated hydroponic culture is very effective for use in genetic analyses of drought tolerance at seedling stage.     

Residual effects of cultivation methods on weed seed banks and weeds in Cambodia

The effects of the planting method (transplanting vs. direct seeding), rice cultivar (Sen Pidao vs. Phka Rumduol), and herbicide application (admixture of bentazone and cyhalofop‐butyl) on weeds and weed seed banks were quantified in unflooded, shallowly flooded, and deeply flooded paddy fields in Cambodia in 2005 and 2006. Broad‐leaved weeds infested more toward maturity in 2006 than in 2005, particularly in directly seeded plots. Weed dry weights at pre‐heading and/or maturity were consistently reduced by herbicide application and Phka Rumduol cultivar, while weed numbers increased under unflooded condition. The proportion of sedges was consistently larger in directly seeded and non‐herbicide plots at pre‐heading. Larger numbers and dry weights of sedges and total weeds in 2005 caused larger seed bank sizes of sedges and total weeds in 2006, which further caused their infestation in 2006. A greater weed dry weight at 62 days after sowing in 2005 resulted in larger seed banks of Cyperus iria and Fimbristylis miliacea, which were most severe under non‐herbicide, direct‐seeded treatment, while that at rice maturity resulted in larger seed banks of Cyperus difformis, Scirpus juncoides, and Lindernia antipoda. Overall, sedges shared the majority of the total weed seed bank, followed by broad‐leaved weeds and then grasses. A lower yield in 2005 led to significantly larger seed bank sizes of sedges, but not of grasses or broad‐leaved weeds. Rice yield reduction was consistently related to larger numbers of sedges by heading and those of grasses at maturity.     

Review of diagnostic plaque reduction neutralization tests for flavivirus infection

Flavivirus infections (including Japanese encephalitis, West Nile encephalitis and dengue fever/severe dengue) present a worldwide public health problem. Recent climate change may affect the geographical distribution of the arthropod vectors for these viruses and so the risk of flavivirus epidemics may increase. Many methods have been developed for the serological diagnosis of flavivirus infections, such as haemagglutination inhibition assay, enzyme-linked immunosorbent assay, and immunofluorescence in staining. However, the specificity of these assays varies.The plaque reduction neutralizing test (PRNT) using live viruses is currently the ‘gold standard’ for the differential serodiagnosis of flaviviruses. The specificity of results obtained with PRNT is better than that for other protocols and many laboratories apply the PRNT protocol to the differential serodiagnosis of flaviviruses. Here, recent refinements to the PRNT protocols with genetically modified recombinant viruses or reporter-harbouring virus-like particles are reviewed. Further, the problems associated with the differential serodiagnosis of flaviviruses using PRNT are discussed.     

Genetically modified rapeseed oil containing cis-9,trans-11,cis-13-octadecatrienoic acid affects body fat mass and lipid metabolism in mice

Punicic acid, one of the conjugated linolenic acid (CLN) isomers, exerts a body-fat reducing effect. Although punicic acid is found in pomegranate and Tricosanthes kirilowii seeds, the amount of this fatty acid is very low in nature. The goal of this study was to produce a transgenic oil containing punicic acid. A cDNA encoding conjugase that converts linoleic acid to punicic acid was isolated from T. kirilowii, and the plant expression vector, pKN-TkFac, was generated. The pKN-TkFac was introduced into Brassica napus by Agrobacterium-mediated transformation. As a result, a genetically modified rapeseed oil (GMRO) containing punicic acid was obtained, although its proportion to the total fatty acids was very low (approximately 2.5%). The effects of feeding GMRO in ICR CD-1 male mice were then examined. Wild-type rapeseed (B. napus) oil (RSO) containing no CLN was used as a control oil. For reference oils, RSO-based blended oils were prepared by mixing with different levels of pomegranate oil (PO), either 2.5% (RSO + PO) or 5.0% (RSO + 2PO) punicic acid. Mice were fed purified diets containing 10% of either RSO, RSO + PO, RSO + 2PO, or GMRO for 4 weeks, and dietary PO dose-dependently reduced perirenal adipose tissue weight with a significant difference between the RSO group and the RSO + 2PO group. GMRO, as compared to RSO, lowered the adipose tissue weight to the levels observed with RSO + 2PO. The liver triglyceride level of the RSO + 2PO and GMRO groups but not that of the RSO + PO group was lower than that of the RSO group. The RSO + 2PO and GMRO groups, but not the RSO + PO group, had increased carnitine-palmitoyltransferase activity in the liver and brown adipose tissue. These results showed that dietary GMRO, even at a dietary punicic acid level as low as 0.25 wt % of diet, reduced body fat mass and altered liver lipid metabolism in mice and was more effective than an equal amount of punicic acid from PO.     

Pluripotency‐associated genes reposition during early embryonic developmental stages in pigs

We examined the allelic expression and positioning of two pluripotency‐associated genes, OCT4 and SOX2, and two housekeeping genes, ACTB and TUBA, in 4‐ and 8‐cell porcine embryos utilizing RNA and DNA fluorescence in situ hybridization (FISH) in single blastomeres. The proportion of blastomeres expressing SOX2 bi‐allelically increased from 45% at the 4‐cell stage to 60% at the 8‐cell stage. Moreover, in 8‐cell embryos, SOX2 was expressed bi‐allelically in significantly more blastomeres than was the case for OCT4, and this was associated with a tendency for SOX2 alleles to move toward the nuclear interior during 4‐ to 8‐cell transition. However, the radial location of OCT4 alleles did not change significantly during this transition. The locations of active and inactive alleles based on DNA and RNA FISH signals were also calculated. Inactive OCT4 alleles were located in very close proximity to the nuclear membrane, whereas active OCT4 alleles were more centrally disposed in the nucleus. Nevertheless, the nuclear location of active and inactive SOX2 alleles did not change in either 4‐ or 8‐cell blastomeres. Our RNA and DNA FISH data provide novel information on the allelic expression patterns and positioning of pluripotency‐associated genes, OCT4 and SOX2, during embryonic genome activation in pigs.     

Quantitative analyses of lymphoid tissue in the spleen, lymph nodes and Peyer's patches in cynomolgus monkeys

To clarify the morphological characteristics of the cynomolgus monkey immune system, we analyzed quantitative data on their lymphoid organs. Spleens, major lymph nodes and Peyer's patches were sampled from cynomolgus monkeys, and the lymphoid follicle and germinal center areas and percentages of CD3- and CD20-positive areas were calculated. All the organs analyzed showed large interindividual variations in the sizes of lymphoid follicles and germinal centers. Lymphoid follicle in the spleen, submandibular lymph nodes and Peyer's patches showed no marked difference in size. Germinal center size in the mesenteric lymph nodes and Peyer's patches were significantly smaller than those in the spleen. Areas containing T cells were largest in the lymph nodes, while those containing B cells were largest in the spleen and Peyer's patches. The mean size of the splenic lymphoid follicle in cynomolgus monkeys is larger than that in rats and similar to that in humans. Based on the large individual variation and the characteristics of lymphoid organs, it is important to use cynomolgus monkeys in standard toxicity studies. Taking advantage of the characteristics of each species enables reliable evaluation of the immunologic system in standard toxicity studies.     

Factors affecting fertilization and embryonic development during intracytoplasmic sperm injection in pigs

In intracytoplasmic sperm injection (ICSI) technique, a sperm was injected into ooplasm directly using a glass pipette. The fertilization physiology in ICSI is considered quite different from that of the natural fertilization. The different mechanisms for fertilization may be the causes of various results in ICSI. In this paper, we focus on the state of sperm membranes, nuclear or DNA integrity during ICSI procedure and discuss the influence of these factors on fertilization and embryonic development. We also introduce some examples in application of ICSI for new technologies in pigs.