Developmental ability of vitrified mouse oocytes expressing water channels

Previously, we showed that the exogenous expression of aquaporin 3 (AQP3), an aquaglyceroporin, improved the tolerance of mouse oocytes to vitrification with a glycerol-based solution. In the present study, we examined conditions suitable for the expression of AQP3 and the ability of vitrified oocytes to develop in vitro and in vivo after fertilization. After only partial remove of cumulus cells, immature mouse oocytes (germinal vesicle stage) were injected with 5, 10 or 20 pg of AQP3 cRNA and cultured for 12 h for maturation. When matured oocytes were vitrified with a glycerol-based solution, 57-61% survived regardless of the amount of cRNA injected (5-20 pg). By contrast, no oocytes injected with water (control) survived. When the zona pellucida was removed from the vitrified oocytes and the oocytes were then fertilized in vitro and cultured, the proportions that were fertilized and developed into blastocysts were higher when the amount of cRNA injected was 5 pg than 10-20 pg. When 16 blastocysts were transferred to a pseudopregnant mouse, 5 developed to term, demonstrating that oocytes vitrified after injection of AQP3 cRNA retained the ability to develop to term. The water-permeability of cRNA-injected oocytes was higher than that of control oocytes from the maturing stage to the 1-cell zygote stage, whereas glycerol-permeability was higher only at metaphase II. This indicates that AQP3 was expressed for a relatively short period of time. These results suggest that the transient expression of water/cryoprotectant channels is effective for cryopreserving cells that have low membrane-permeability, such as mammalian oocytes.     

Food sources for the bivalve Corbicula japonica in the foremost fishing lakes estimated from stable isotope analysis

ABSTRACT:   Carbon and nitrogen stable isotope ratios in tissue of the bivalve corbicula Corbicula japonica and particulate organic matter (POM) were measured in Lake Jusan, Lake Ogawara and Lake Shinji, which are the foremost fishing grounds for the corbicula in Japan, to determine their food sources. The bivalves in Lake Ogawara and Lake Shinji showed enriched isotope composition, while those in Lake Jusan were depleted. In addition, the difference in the isotope ratios between the sampling sites was remarkable in Lake Jusan. Chlorophyll concentrations were significantly higher in Lake Ogawara and Lake Shinji than those in the inflow rivers, although that in Lake Jusan was equivalent to that in the river. Residence time of river water was estimated at 1 day, 455 days and 88 days in Lake Jusan, Lake Ogawara and Lake Shinji, respectively. These values indicate that the bivalves in Lake Ogawara and Lake Shinji assimilate autochthonous phytoplankton, while those in Lake Jusan assimilate terrestrial matter in the upper reaches and marine phytoplankton in the lower reaches because of low production in the lake.     

Morphological reproductive characteristics of testes and fertilization capacity of cryopreserved sperm after the Fukushima accident in raccoon (Procyon lotor)

Since the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident, we have established an archive system of livestock and wild animals from the surrounding ex-evacuation zone. Wildlife within the alert zone have been exposed to low-dose-rate (LDR) radiation for a long continuous time. In this study, we analysed the morphological characteristics of the testes and in vitro fertilization (IVF) capacity of cryopreserved sperm of racoons from the ex-evacuation zone of the FDNPP accident. The radioactivity of caesium-137 (¹³⁷Cs) was measured by gamma-ray spectrometry, and the measured radioactivity concentration was 300–6,630 Bq/kg in the Fukushima raccoons. Notably, normal spermatogenesis was observed in the seminiferous tubules of the testes, with the germinal epithelium composed of a spermatogenic cell lineage with no evident ultrastructural alterations; freeze-thawing sperm penetration ability was confirmed using the interspecific zona pellucida-free mouse oocytes IVF assays. This study revealed that the chronic and LDR radiation exposure associated with the FDNPP accident had no adverse effect on the reproductive characteristics and functions of male raccoons.     

Detection of giant myofibrillar proteins connectin and nebulin in fish meat by electrophoresis in 3-5 gradient sodium dodecyl sulfate polyacrylamide slab gels

An improved method was investigated for sodium dodecyl sulfate polyacrylamide slab gel electrophoresis (SDS-PAGE) to facilitate the analysis of the giant myofibrillar proteins, connectin and nebulin, in fish meat by using jack mackerel (Trachurus japonicus) as the sample fish. It was established that separation of the alpha-connectin band from the beta-connectin band by SDS-PAGE could be achieved by using 3-5% gradient gels with glycerol to facilitate the formation of a gradient with polymerization at 35 degrees C. SDS-PAGE samples of white dorsal muscle from the jack mackerel were homogenized with a 2% SDS solution containing an inhibitor mixture (1 microg/mL of phenylmethanesulfonyl fluoride, 1 microg/mL of leupeptin, and 1 microg/mL of E-64) and heated at 50 degrees C for 20 min. Heating these samples at 100 degrees C for 2 min resulted in the disintegration of connectin but did not affect nebulin. A purified myofibril sample and a whole muscle sample showed similar changes in the overall rate of degradation of whole connectin and nebulin during the postmortem storage period, but it was clear that beta-connectin was cleaved from alpha-connectin during the preparation of myofibrils at the early stage postmortem. Storage of the SDS-PAGE samples at -85 degrees C was preferable to storage at -18 degrees C for a long period.     

Enzymatic high digestion of soybean milk residue (okara)

The objective of this study was to digest okara in high yield by food-processing enzymes. Autoclaving of okara was effective in increasing cellulase digestion for the primary cell wall, and the digestion was accelerated by the formation of single cells by stirring. Most of the residual okara after autoclaving and cellulase digestion was found to be the secondary cell walls compared with the cellulase-treated soybean single cells. The secondary cell wall was found to be composed of galacturonic acid, neutral sugars, and protein and was considered to be a complex of these compositions. Many cellulolytic and proteolytic enzymes could not digest the secondary cell wall; however, it was found that two pectinases could digest the secondary cell wall. A series of digestions resulted in yields of 83-85% from the raw okara, and the final residues were identified as oil body complexes in the soybean cells and fiber-like organ between the cells.     

Determination and Speciation of Aluminum in Environmental Samples by Cation Exchange High-Performance Liquid Chromatography with High Resolution ICP-MS Detection

A cation-exchange high-performance liquid chromatography with high resolution inductively coupled plasma mass spectrometric detection (CE-HPLC/ICP-MS) was developed for the determination and the speciation of aluminum in environmental samples. Three types of aluminum species (AlL_x ^<+2, AlL_x ²⁺, Al³⁺) were separated from one another, and were determined with the present system. The comparison of the present system with an established CE-HPLC with fluorimetric detection using 5-sulfo-8-quinolinol (CE-HPLC/FL) was described. The present system showed better sensitivity for aluminum than CE-HPLC/FL. Moreover, the analytical results for soil extract and lake water samples obtained with both methods were in good agreement with each other.     

Antioxidant levels in watercore tissue in ‘Fuji’ apples during storage

Watercore usually spreads in mature ‘Fuji’ apples and may induce flesh browning disorders through unknown mechanisms. We analyzed H₂O₂ and ascorbic acid (AA) contents and the activities of four enzymes involved in the AA–glutathione (AA–GSH) cycle in watercore and watercore-free tissues in ‘Fuji’ apple fruit during storage. H₂O₂ levels always were higher in watercore than in watercore-free tissues. AA levels were lower in watercore tissues at harvest and decreased in both tissue types during the storage period. However, AA was completely absent from watercore tissue but not watercore-free tissue three months after harvest. Ascorbate peroxidase (APX) activities were always higher in watercore than in watercore-free tissues, and the activities of dehydroascorbate reductase (DHAR) decreased continuously after harvest in both tissue types. These results suggested that higher production of H₂O₂ caused by anaerobic conditions in watercore activated APX which acted as a redox signal; the concomitant net consumption of AA was not balanced by the decline of DHAR activity, leading to decreased antioxidant levels. On the other hand, the gradual increases in monodehydroascorbate reductase (MDAR) and glutathione reductase (GR) activity observed during storage accompanied by low AA levels and DHAR activity might indicate a declining efficiency of the AA–GSH cycle.     

Size-exclusion chromatography of tea tannins and intercepting potentials of peptides for the inhibition of trypsin-caseinolytic activity by tea tannins

Molecular-weight distribution and characterization of tea tannin were investigated by high-performance liquid chromatography and the equivalent preparative exclusion gel chromatography using Sephadex G-25. The characteristics of the fractions were studied regarding the amounts of terminal catechin, sugar, and gallic acid, the color reaction of the Folin-Chiocalteu reagent, the UV absorbance, and the inhibition activity for the trypsin-caseinolytic activity per weight. Furthermore, we investigated the intercepting activities of the inhibition by the amino acids, peptides, their analogues, poly(ethylene glycol)s (PEGs), and histatin 5 using the inhibition of trypsin-caseinolytic activity by tea. Arg, Lys, and their peptides had strong intercepting activities for the inhibition, but only a weak activity was detected in the Pro peptides or gelatin-like peptides of (Pro-Pro-Gly)(n) (n = 5 or 10). The guanidyl group of Arg and the amino methylene group of Lys were important for the intercepting activity, but the activity was weakly dependent upon the peptide bond formation. The intercepting activity of the peptides or PEG exponentially increased with the number of polymerizations. Histatin 5 did not have a remarkably strong intercepting activity considering the peptide length. The activity of the synthetic histatin 5 in which all of the Lys and Arg were substituted by Ala was at the same level as histatin 5.     

Two-week Toxicity of Multi-walled Carbon Nanotubes by Whole-body Inhalation Exposure in Rats

To evaluate pulmonary toxicity of multi-walled carbon nanotubes (MWCNTs), F344 rats of both sexes were exposed by inhalation to 0.2, 1 or 5 mg/m³ MWCNT aerosol for 6 h/day, 5 days/week for 2 weeks using a whole-body exposure system. At the end of the 2-week exposure period, one-half of the rats were necropsied, and at the end of an additional 4-week postexposure period, the remaining rats were necropsied. MWCNTs were deposited in the lungs of all MWCNT-exposed groups and mostly remained in the lungs throughout the 4-week postexposure period. Granulomatous changes in the lung were found in the rats exposed to 5 mg/m³ MWCNTs, and these changes were slightly aggravated at the end of the 4-week postexposure period. In the bronchoalveolar lavage fluid (BALF), the numbers of neutrophils, percentages of bi- and multinucleated alveolar macrophages, levels of ALP activity and concentrations of total protein and albumin were elevated in the rats exposed to 1 and 5 mg/m³ MWCNTs. At the end of the 4-week postexposure period, the values of the BALF parameters tended to remain elevated. In addition, goblet cell hyperplasias in the nasal cavity and nasopharynx were observed in the rats exposed to 1 and 5 mg/m³ MWCNTs, but these lesions had largely regressed by the end of the postexposure period. Based on the histopathological and inflammatory changes, the no-observed-adverse-effect level (NOAEL) for inhalation of MWCNTs for 2 weeks was 0.2 mg/m³.     

Properties of L-type bovine spongiform encephalopathy in intraspecies passages

The origin and transmission routes of atypical bovine spongiform encephalopathy (BSE) remain unclear. To assess whether the biological and biochemical characteristics of atypical L-type BSE detected in Japanese cattle (BSE/JP24) are conserved during serial passages within a single host, 3 calves were inoculated intracerebrally with a brain homogenate prepared from first-passaged BSE/JP24-affected cattle. Detailed immunohistochemical and neuropathologic analysis of the brains of second-passaged animals, which had developed the disease and survived for an average of 16 months after inoculation, revealed distribution of spongiform changes and disease-associated prion protein (PrP(Sc)) throughout the brain. Although immunolabeled PrP(Sc) obtained from brain tissue was characterized by the presence of PrP plaques and diffuse synaptic granular accumulations, no stellate-type deposits were detected. Western blot analysis suggested no obvious differences in PrP(Sc) molecular mass or glycoform pattern in the brains of first- and second-passaged cattle. These findings suggest failures to identify differences in mean incubation period and biochemical and neuropathologic properties of the BSE/JP24 prion between the first and second passages in cattle.