Copy number-dependent DNA methylation of the Pyricularia oryzae MAGGY retrotransposon is triggered by DNA damage.

Transposable elements are common targets for transcriptional and post-transcriptional gene silencing in eukaryotic genomes. However, the molecular mechanisms responsible for sensing such repeated sequences in the genome remain largely unknown. Here, we show that machinery of homologous recombination (HR) and RNA silencing play cooperative roles in copy number-dependent de novo DNA methylation of the retrotransposon MAGGY in the fungus Pyricularia oryzae. Genetic and physical interaction studies revealed that RecA domain-containing proteins, including P. oryzae homologs of Rad51, Rad55, and Rad57, together with an uncharacterized protein, Ddnm1, form complex(es) and mediate either the overall level or the copy number-dependence of de novo MAGGY DNA methylation, likely in conjunction with DNA repair. Interestingly, P. oryzae mutants of specific RNA silencing components (MoDCL1 and MoAGO2) were impaired in copy number-dependence of MAGGY methylation. Co-immunoprecipitation of MoAGO2 and HR components suggested a physical interaction between the HR and RNA silencing machinery in the process.

Effects of methotrexate exposure on medaka (Oryzias latipes) testes.

For the purpose of clarifying the histopathological effects of methotrexate (MTX) on medaka testes, wild-type and homogenic p53-deficient male medaka at 4 to 6 months post-hatching were exposed to 0.25 mg/ml of MTX for 96 h with histopathological examination of testes at 24, 48, 72 and 96 h. At 72 and 96 h after the start of MTX exposure, numerous apoptotic cells were observed in the spermatogonia and spermatocytes, and the pyknotic cell rate and the TUNEL-positive and cleaved caspase-3-positive rates in the spermatogonia and spermatocytes of MTX-treated wild type medaka were higher compared with those in the control wild-type medaka. Starting at 48 h, the phospho-histone H3-positive rate in the spermatogonia and spermatocytes of was significantly lower in MTX-treated wild-type medaka than in control wild-type medaka. In homogenic p53-deficient medaka, apoptosis was not induced in the spermatogonia and spermatocytes by exposure to MTX. Starting at 48 h, the phospho-histone H3-positive rate in spermatogonia and spermatocytes of MTX-treated homogenic p53-deficient medaka was lower than in control homogenic p53-deficient medaka. Throughout the entire experimental period, there were no significant differences in phospho-histone H3-positive rates in the spermatogonia and spermatocytes between the MTX-treated homogenic p53-deficient medaka group and the MTX-treated wild-type medaka group. In the present study, spermatogonia and spermatocytes of medaka testes were sensitive to MTX at 0.25 mg/ml in the culture water, and MTX-induced apoptosis in the testes was dependent on p53 expression; however, inhibition of MTX-induced cell proliferation was independent of p53 expression.

Effects of cadmium exposure on Iberian ribbed newt (Pleurodeles waltl) testes.

To characterize the histomorphologic effects of cadmium on adult newt testes, male Iberian ribbed newts (6 months post-hatching) were intraperitoneally exposed to a single dose of 50 mg/kg of cadmium, with histologic analysis of the testes at 24, 48, 72, and 96 h. Beginning 24 h after cadmium exposure, apoptosis of spermatogonia and spermatocytes was observed, and congestion was observed in the interstitial vessels of the testes. Throughout the experimental period, the rates of pyknotic cells and TUNEL and cleaved caspase-3 positivity were significantly higher in the spermatogonia and spermatocytes of cadmium-treated newts compared with control newts. There were no significant differences between cadmium-treated and control newts in phospho-histone H3 positivity in the spermatogonia and spermatocytes. These results suggest that spermatogonia and spermatocytes in adult Iberian ribbed newts are highly sensitive to cadmium. This is the first report of the histomorphologic characteristics of cadmium-induced testicular dysfunction in newts.

Effects of cadmium exposure on medaka (Oryzias latipes) testes.

Adult male medaka (Oryzias latipes) were exposed to 10 ppm of cadmium for 96 h, and the testes were examined histopathologically. Numerous apoptotic cells were found in the spermatogonia and spermatocytes at 72 and 96 h after initiation of cadmium exposure, and the pyknotic index, TUNEL-positive rate, and cleaved caspase-3-positive rate in the spermatogonia and spermatocytes of the cadmium-treated group were higher compared with the control group. No significant difference between the control and cadmium-treated groups was found in the phospho-histone H3-positive rate in the spermatogonia and spermatocytes. No edematous, hemorrhagic, or necrotic changes were observed within the testes in the cadmium-treated group. These results suggest that spermatogonia and spermatocytes in medaka testes are highly sensitive to cadmium. Exposure to 10 ppm of cadmium induced histopathologic changes in the testes that were similar to those described in rodents exposed to low doses of cadmium.

Double-knockout of putative endo-ß-N-acetylglucosaminidase (ENGase) genes in Arabidopsis thaliana: loss of ENGase activity induced accumulation of high-mannose type free N-glycans bearing N,N'-acetylchitobiosyl unit.

Endo-ß-N-acetylglucosaminidase (ENGase) is involved in the production of high-mannose type free N-glycans during plant development and fruit maturation. In a previous study (K. Nakamura et al. Biosci. Biotechnol. Biochem., 73, 461-464 (2009)), we identified the tomato ENGase gene and found that gene expression remained relatively constant. In the present study, we constructed an Arabidopsis thaliana mutant in which the expression of two putative ENGase genes was suppressed. The mutant showed no ENGase activity, but produced high-mannose type free N-glycans carrying the N,N'-acetylchitobiosyl unit that is produced by peptide:N-glycanase, indicating that both these genes encode Arabidopsis ENGase.