Seasonal changes in oocyte size and maturity of the giant jellyfish, Nemopilema nomurai

To determine the reproductive season of the giant jellyfish Nemopilema nomurai, we investigated gonadal maturity in specimens collected from the East China Sea, Korea Strait, Wakasa Bay, and the Shonai Coast of Yamagata Prefecture. After the sex of the samples was determined, the long axis of at least 256 oocytes from each female was measured. In specimens collected from the coast of Japan in 2005 and in 2006, all gonads were sufficiently developed to determine sex. However, 18 of the 20 specimens from the East China Sea collected in July 2005 were immature, and sex could not be determined. The maximum and third quartile of oocyte length had a significant correlation with days elapsed from 30 June, but they were not related to bell diameter. Observations of gonad tissue sections of specimens collected in Wakasa Bay in 2006 confirmed that oocyte length was a good proxy for female maturity. Male maturity could also be determined. In conclusion, the sex of all of the small-sized medusae collected along the coast of Japan was determinable, and their gonads were at various stages of development up to fully mature. Therefore, the occurrence of small-sized jellyfish during the autumn in Wakasa Bay is not caused by recruitment of young population from the nearby coast.     

Neuronal activation modulates enhancer activity of genes for excitatory synaptogenesis through de novo DNA methylation

Post-mitotic neurons do exhibit DNA methylation changes, contrary to the longstanding belief that the epigenetic pattern in terminally differentiated cells is essentially unchanged. While the mechanism and physiological significance of DNA demethylation in neurons have been extensively elucidated, the occurrence of de novo DNA methylation and its impacts have been much less investigated. In the present study, we showed that neuronal activation induces de novo DNA methylation at enhancer regions, which can repress target genes in primary cultured hippocampal neurons. The functional significance of this de novo DNA methylation was underpinned by the demonstration that inhibition of DNA methyltransferase (DNMT) activity decreased neuronal activity-induced excitatory synaptogenesis. Overexpression of WW and C2 domain-containing 1 (Wwc1), a representative target gene of de novo DNA methylation, could phenocopy this DNMT inhibition-induced decrease in synaptogenesis. We found that both DNMT1 and DNMT3a were required for neuronal activity-induced de novo DNA methylation of the Wwc1 enhancer. Taken together, we concluded that neuronal activity-induced de novo DNA methylation that affects gene expression has an impact on neuronal physiology that is comparable to that of DNA demethylation. Since the different requirements of DNMTs for germ cell and embryonic development are known, our findings also have considerable implications for future studies on epigenomics in the field of reproductive biology.     

Single spaghetti tagging as a high-retention marking method for Japanese common sea cucumber <Emphasis Type="Italic">Apostichopus japonicus</Emphasis>

Populations of sea cucumbers, including the Japanese common sea cucumber Apostichopus japonicus, have been seriously depleted worldwide due to overfishing. Mark–recapture study is an efficient means of collecting ecological data. However, the use of such a method in sea cucumbers is difficult because they lack hard tissues in the body wall. Here we tested the viability of various tagging methods on A. japonicus. First, we applied conventional tags using four different methods [single spaghetti (T-bar) tagging, double spaghetti tagging, ribbon tagging, and Atkins tagging] to ten individuals per method in aquaria for 14 days. Of the methods used, single spaghetti tagging had the highest retention rate. Then we examined the retention rate of single spaghetti tags on ten individual sea cucumbers for up to approximately 6 months in rearing conditions. The single spaghetti tagging method showed a retention rate of 100% over at least 7 days, and 50% of the tags remained embedded after 56 days. The longest duration of tag retention was 174 days, at which time the experiment was terminated. These results indicate that single spaghetti tagging is reliable for both short- and longer-term studies, making it a useful tool for ecological and conservation studies in sea cucumbers.     

Effects of the preservation medium and storage duration of domestic cat ovaries on the maturational and developmental competence of oocytes in vitro

We examined the effectiveness of saline, Euro-Collins solution (EC), and ET-Kyoto solution (ET-K) as preservation media for the cold storage of feline ovaries. Ovaries were maintained in these media at 4°C for 24, 48, or 72 h until oocyte retrieval. The ET-K group exhibited a higher oocyte maturation rate than the saline group after 72 h of storage. Moreover, ET-K could sustain the competence of the feline oocytes to cleave after 48 h, and the morula formation rate of the ET-K group was higher than that of the other groups after 24 and 48 h. Furthermore, the ET-K group exhibited a higher blastocyst formation rate than the other groups after storage for 24 h, and only ET-K retained the developmental competence in blastocysts after 48 h of storage. In addition, regarding the cell numbers of the blastocysts, there was no significant difference among the tested groups. In conclusion, our results indicate that ET-K is a suitable preservation medium for feline ovaries.     

Evaluation of a d-Octaarginine-linked polymer as a transfection tool for transient and stable transgene expression in human and murine cell lines

Poly(N-vinylacetamide-co-acrylic acid) coupled with d-octaarginine (VP-R8) promotes the cellular uptake of peptides/proteins in vitro; however, details of the transfection efficacy of VP-R8, such as the cell types possessing high gene transfer, are not known. Herein, we compared the ability of VP-R8 to induce the cellular uptake of plasmid DNA in mouse and human cell lines from different tissues and organs. A green fluorescent protein (GFP)-expression plasmid was used as model genetic material, and fluorescence as an indicator of uptake and plasmid-derived protein expression. Three mouse and three human cell lines were incubated with a mixture of plasmid and VP-R8, and fluorescence analysis were performed two days after transfection. To confirm stable transgene expression, we performed drug selection three days after transfection. A commercially available polymer-based DNA transfection reagent (PTR) was used as the transfection control and standard for comparing transgene expression efficiency. In the case of transient transgene expression, slight-to-moderate GFP expression was observed in all cell lines transfected with plasmid via VP-R8; however, transfection efficiency was lower than using the PTR for gene delivery. In the case of stable transgene expression, VP-R8 promoted drug-resistance acquisition more efficiently than the PTR did. Cells that developed drug resistance after VP-R8-mediated gene transfection expressed GFP more efficiently than cells that developed drug resistance after transfection with the PTR. Thus, VP-R8 shows potential as an in vitro or ex vivo nonviral transfection tool for generating cell lines with stable transgene expression.     

Inclusion body hepatitis caused by fowl adenovirus in broiler chickens in Japan, 2009-2010

From January 2009 to June 2010, many broiler chicks suddenly died without clinical signs. The mortality rates were from 1.2% to 17.0% in affected flocks. Inclusion body hepatitis (IBH) was detected in 13 prefectures (northern, eastern, western, and southern areas) in Japan. The livers were enlarged and pale. The bursa of Fabricius and thymus had not atrophied. Multifocal necroses of hepatocytes with basophilic intranuclear inclusions were seen in the liver. Eosinophilic intranuclear inclusion bodies in hepatocytes were rare. Focal necrosis of acinar cells with basophilic intranuclear inclusions was found in the pancreas. Basophilic intranuclear inclusion bodies were detected in intact surface epithelial cells of gizzard and epithelial cells of the small intestine. The intranuclear inclusions of liver, pancreas, gizzard, and small intestine were stained positively for immunohistochemistry of fowl adenovirus (FAV) antigen. Ultrastructurally, basophilic intranuclear inclusions consisted of viral particles approximately 70 nm in diameter and arranged in a crystalline array. FAV was isolated from the liver of chickens affected with IBH. The serotype of most isolates was 2. This study suggests that IBH produced by FAV is epidemic in broiler chicks in Japan and that the present cases occurred as the primary disease without the association of infectious bursal disease virus or chicken anemia virus.     

Immunological characterization of peripheral blood leukocytes using vaccine for mycoplasmal pneumonia of swine (MPS) in swine line selected for resistance to MPS

This study was conducted to evaluate immunological changes in peripheral blood leukocytes in pigs that were genetically selected for their improved resistance to mycoplasmal pneumonia of swine (MPS), using MPS vaccine as an antigen. Twelve castrated MPS‐selected Landrace pigs were compared with the same number of pigs from a nonselected line by using a time‐course analysis at the hematological level. After the second sensitization with MPS vaccine, the percentages of B cells, CD4⁺ T cells, and natural killer (NK) cells in total leukocytes were lower in the selected line than in the nonselected line, whereas the percentage of granulocytes in total leukocytes increased in the MPS‐selected line. We also assessed the proliferative ability of peripheral blood mononuclear cells (PBMCs) stimulated with Mycoplasma hyopneumoniae, lipopolysaccharide or concanavalin A, and found that although the proliferative ability of the PBMC was not different between the two lines at a steady state, the nonselected line showed a significantly higher proliferative ability after sensitization with MPS vaccine than the selected line regardless of antigens used. These results thus indicate that the selection of pigs on the basis of MPS resistance changes their immunophenotype, and would give us beneficial information for the prevention of MPS infection.     

Analysis of antibody response by temperature-sensitive measles vaccine strain in the cotton rat model

Measles virus (MeV) vaccine strain, AIK-C, is temperature sensitive (ts), which is thought to be associated with attenuation of virus pathogenicity. In this study, replication and antibody response were examined in cotton rats using viruses carrying different forms of the P gene, which is responsible for the ts phenotype of strain AIK-C and its parental Edmonston strain. When cotton rats were inoculated intranasally, ts viruses neither replicated in lungs, nor reproducibly generated an antibody response. When inoculated intramusculary (i.m.), however, ts strains raised an antibody titer in all animals. This response was not observed when ultraviolet-inactivated virus was used. ts virus, inoculated i.m., was recovered from cotton rat drainage lymph nodes. These results suggest that ts virus, inoculated i.m., could replicate in the cotton rat, presumably at the superficial lymph node, and induce an antibody response. Therefore, cotton rats can serve as a small-animal model for investigating immune responses to safer ts vaccine, as well as recombinant vaccine using AIK-C as a vector for protection against other infectious agents.